Antisense modulation of transforming growth factor-beta expression

ABSTRACT

Compositions and methods are provided for inhibiting the expression of TGF-β2. Antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding TGF-β2 are preferred. Methods of using these compounds for modulation of TGF-β2 expression and for treatment of diseases associated with expression of TGF-β are also provided.

INTRODUCTION

[0001] This application is a continuation of U.S. application Ser. No.09/948,002 filed Sep. 5, 2001, which is a continuation-in-part of U.S.application Ser. No. 09/661,753, filed Sep. 14, 2000 issued as U.S. Pat.No. 6,436,909, which claims the benefit of U.S. Provisional ApplicationNo. 60/154,546 filed Sep. 17, 1999.

FIELD OF THE INVENTION

[0002] The present invention provides compositions and methods formodulating the expression of transforming growth factor-β (TGF-β). Inparticular, this invention relates to antisense compounds, particularlyoligonucleotides, specifically hybridizable with nucleic acids encodinghuman TGF-β. Such oligonucleotides have been shown to modulate theexpression of TGF-β.

BACKGROUND OF THE INVENTION

[0003] Transforming growth factor-β (TGF-β) is a cytokine whichregulates biological processes such as cell proliferation,differentiation and immune reaction. It has been found to have manyactions in tissue repair, stimulating the deposition of extracellularmatrix in multiple ways. TGF-β stimulates the synthesis of matrixproteins including fibronectin, collagens and proteoglycans. It alsoblocks the degradation of matrix by inhibiting protease secretion and byinducing the expression of protease inhibitors. It also facilitatescell-matrix adhesion and matrix deposition via modulation of expressionof integrin matrix receptors, and lastly TGF-β also upregulates its ownexpression. TGF-β exists in three isoforms in mammals: TGF-β1, -2 and-3. These function similarly in vitro.

[0004] Fibrosis is a pathological process, usually resulting frominjury, which can occur in any organ. Excessive amounts of extracellularmatrix accumulate within a tissue, forming scar tissue which causesdysfunction and, potentially, organ failure. Fibrosis can be eitherchronic or acute. Chronic fibrosis includes fibrosis of the majororgans, most commonly liver, kidney and/or heart, and normally has agenetic or idiopathic origin. Progressive fibrosis of the kidney is themain cause of chronic renal disease. In diabetics, fibrosis withinglomeruli (glomerulosclerosis) and between tubules (tubulointerstitialfibrosis) causes the progressive loss of renal function that leads toend-stage renal disease. Fibrotic lung disorders include some 180different conditions and result in severe impairment of lung function.

[0005] Acute fibrosis is associated with injury, often as a result ofsurgery. Surgical adhesion represents the largest class of acutefibrosis. Surgery often results in excessive scarring and fibrousadhesions. It is estimated that over 90% of post-surgical patients areaffected by adhesions. Abdominal adhesions can lead to small bowelobstruction and female infertility. Fibrosis after neck and back surgery(laminectomy, discectomy) can cause significant pain. Fibrosis after eyesurgery can impair vision. Pericardial adhesions after coronary bypasssurgery, fibrosis after organ transplant rejection and general scarringafter plastic surgery are other examples. This represents a major unmetmedical need.

[0006] Antisense and other inhibitors of TGF-β have been used toelucidate the role of TGF-βs in cancer, anaphylaxis, fibrosis and otherconditions. As examples:

[0007] Dzau (WO 94/26888) discloses use of antisense sequences whichinhibit the expression of cyclins and growth factors including TGF-β₁,TGF, bFGF, PDGF for inhibiting vascular cellular activity of cellsassociated with vascular lesion formation in mammals. Shen et al.discloses use of phosphorothioate antisense oligonucleotides targeted toTGF-β2 to reduce TGF-β2 expression in U937 cells (Bioorg. Med. Chem.Lett., 1999, 9, 13-18).

[0008] Schuftan et al. (1999, Eur. J. Clin Invest., 29, 519-528)disclose use of a2-macroglobin or antisense to TGF-β1 to reduceextracellular matrix synthesis in cultured rat hepatic stellate cells.

[0009] Kim et al. have used antisense oligonucleotides targeted toTGF-β1 to inhibit passive cutaneous anaphylaxis and histamine release.1999, J. Immunol. 162, 4960-4965.

[0010] Kim et al. have also used an antisense TGF-β1oligodeoxynucleotide to inhibit wound-induced expression of TGF-β1 mRNAin mouse skin. Pharmacol. Res., 1998, 37, 289-293.

[0011] Liu et al. used TGF-β antibody or antisense to TGF-β1 to inhibitsecretion of plasminogen activator inhibitor-1 in EGR-1 regulated cells.1999, J. Biol. Chem. 274, 4400-4411.

[0012] Arteaga et al. used antibodies or antisense oligonucleotidestargeted to TGF-β2 to enhance sensitivity of cancer cells to NK cells inthe presence of tamoxifen. 1999, J. Nat. Cancer Inst. 91, 46-53.

[0013] Tzai et al., 1998, Anticancer Res., 18, 1585-1589, used antisenseoligonucleotides specific for TGF-β1 to inhibit in vitro and in vivogrowth of murine bladder cancer cells.

[0014] The role of TGF-β in diabetic nephropathy is reviewed in Hoffman,et al., 1998, Electrolyte Metab., 24, 190-196.

[0015] Neutralizing anti-TGF-β antibodies or antisense oligonucleotidesdirected to TGF-β1 are reported to prevent the hypertrophic effects ofhigh glucose and the stimulation of matrix synthesis in renal cells.

[0016] Antisense phosphorothioate oligodeoxynucleotides targeted toTGF-β3 were used by Nakajima et al. (1998, Japan. Dev. Biol, 194,99-113; abstract only) and others to block transformation ofatrioventricular canal endothelial cells into invasive mesenchyme.

[0017] Chung et al. (U.S. Pat. No. 5,683,988) disclose and claimparticular antisense oligodeoxynucleotides targeted to TGF-β and use ofthese to inhibit scarring.

SUMMARY OF THE INVENTION

[0018] The present invention is directed to antisense compounds,particularly oligonucleotides, which are targeted to a nucleic acidencoding TGF-β, and which modulate the expression of TGF-β.Pharmaceutical and other compositions comprising the antisense compoundsof the invention are also provided. Further provided are methods ofmodulating the expression of TGF-β in cells or tissues comprisingcontacting said cells or tissues with one or more of the antisensecompounds or compositions of the invention. Further provided are methodsof treating an animal, particularly a human, suspected of having orbeing prone to a disease or condition associated with expression ofTGF-β by administering a therapeutically or prophylactically effectiveamount of one or more of the antisense compounds or compositions of theinvention.

[0019] One embodiment of the present invention is a compound 8 to 50nucleobases in length targeted to a nucleic acid molecule encodingTGF-β2 which comprises at least an 8 nucleobase portion of SEQ ID NO:53, 54, 55, 56, 58, 60, 61, 62, 63, 64, 65 or 66 and which modulates theexpression of TGF-β2. Preferably, the compound is an antisenseoligonucleotide. In one aspect of this preferred embodiment, theantisense oligonucleotide comprises at lest one modified internucleosidelinkage. Advantageously, the modified internucleoside linkage is aphosphorothioate linkage. Preferably, the antisense oligonucleotidecomprises at least one modified sugar moiety. In one aspect of thispreferred embodiment, the modified sugar moiety is a 2′-O-methoxyethylsugar moiety. Advantageously, the antisense oligonucleotide comprises atleast one modified nucleobase. Preferably, the modified nucleobase is a5-methylcytosine. In one aspect of this preferred embodiment, theantisense oligonucleotide is a chimeric oligonucleotide.

[0020] The present invention also provides a composition comprising thecompound described above and a pharmaceutically acceptable carrier ordiluent. Advantageously, the composition further comprises a colloidaldispersion system. Preferably, the compound is an antisenseoligonucleotide.

[0021] Another embodiment of the present invention is a method ofinhibiting the expression of TGF-β2 in cells or tissues comprisingcontacting the cells or tissues with the compound described above sothat expression of TGF-β2 is inhibited.

[0022] The present invention also provides a method of treating ananimal having a disease or condition associated with TGF-β2 comprisingadministering to the animal a therapeutically or prophylacticallyeffective amount of the compound described above so that expression ofTGF-β2 is inhibited. Advantageously, the disease or condition isinflammation. Preferably, the disease or condition is fibrosis or afibrotic disease or condition. In one aspect of this preferredembodiment, the fibrotic disease or condition is fibrotic scarring,peritoneal adhesions, lung fibrosis or conjunctival scarring.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a Kaplan-Meier bleb survival curve in rabbits subjectedto glaucoma drainage surgery and treatment with antisenseoligonucleotides to TGF-β2, TGF-βIIR, TGF-β1 and connective tissuegrowth factor (CTGF).

[0024]FIG. 2 is a graph showing anterior chamber depth after glaucomasurgery in rabbits after treatment with antisense oligonucleotides toTGF-β2, TGF-βIIR, TGF-β1 and CTGF.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding TGF-β, ultimately modulating the amountof TGF-β produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodingTGF-β. As used herein, the terms “target nucleic acid” and “nucleic acidencoding TGF-β” encompass DNA encoding TGF-β, RNA (including pre-mRNAand mRNA) transcribed from such DNA, and also cDNA derived from suchRNA. The specific hybridization of an oligomeric compound with itstarget nucleic acid interferes with the normal function of the nucleicacid. This modulation of function of a target nucleic acid by compoundswhich specifically hybridize to it is generally referred to as“antisense”. The functions of DNA to be interfered with includereplication and transcription. The functions of RNA to be interferedwith include all vital functions such as, for example, translocation ofthe RNA to the site of protein translation, translation of protein fromthe RNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The overall effect of such interference with target nucleic acidfunction is modulation of the expression of TGF-β. In the context of thepresent invention, “modulation” means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene product. In thecontext of the present invention, inhibition is a preferred form ofmodulation of gene expression and mRNA is a preferred target. Further,since many genes (including TGF-β) have multiple transcripts,“modulation” also includes an alteration in the ratio between geneproducts, such as alteration of mRNA splice products.

[0026] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding TGF-β. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding TGF-β, regardless of the sequence(s) of such codons.

[0027] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0028] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0029] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0030] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0031] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or in the case of in vitro assays, under conditions in whichthe assays are performed.

[0032] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0033] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotides have been safely and effectively administered to humansand numerous clinical trials are presently underway. It is thusestablished that oligonucleotides can be useful therapeutic modalitiesthat can be configured to be useful in treatment regimes of cells,tissues and animals, especially humans. In the context of thisinvention, the term “oligonucleotide” refers to an oligomer or polymerof ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimeticsthereof. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0034] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about30 nucleobases. Particularly preferred are antisense oligonucleotidescomprising from about 8 to about 30 nucleobases (i.e. from about 8 toabout 30 linked nucleosides). As is known in the art, a nucleoside is abase-sugar combination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2′-, 3′- or 5′-hydroxyl moiety of the sugar. In formingoligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turnthe respective ends of this linear polymeric structure can be furtherjoined to form a circular structure. However, open linear structures aregenerally preferred. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside backboneof the oligonucleotide. The normal linkage or backbone of RNA and DNA isa 3′-5′ phosphodiester linkage.

[0035] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0036] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included.

[0037] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference.

[0038] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

[0039] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

[0040] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0041] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0042] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)CH₃)]₂, where n and m are from 1to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes an alkoxyalkoxy group, 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504). A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE.

[0043] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference.

[0044] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in The Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613, and those disclosed bySanghvi, Y. S., Crooke, S. T., and Lebleu, B. eds., Antisense Researchand Applications, CRC Press, Boca Raton, 1993, pp. 289-302. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

[0045] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121;5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is hereinincorporated by reference.

[0046] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

[0047] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,each of which is herein incorporated by reference.

[0048] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Cleavage of the RNAtarget can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0049] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, each of which is herein incorporated by reference.

[0050] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0051] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules.

[0052] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0053] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0054] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 or in WO 94/26764.

[0055] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0056] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred addition saltsare acid salts such as the hydrochlorides, acetates, salicylates,nitrates and phosphates. Other suitable pharmaceutically acceptablesalts are well known to those skilled in the art and include basic saltsof a variety of inorganic and organic acids, such as, for example, withinorganic acids, such as for example hydrochloric acid, hydrobromicacid, sulfuric acid or phosphoric acid; with organic carboxylic,sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, forexample acetic acid, propionic acid, glycolic acid, succinic acid,maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malicacid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaricacid, glucuronic acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoicacid, 2-acetoxybenzoic acid, embolic acid, nicotinic acid orisonicotinic acid; and with amino acids, such as the 20 alpha-aminoacids involved in the synthesis of proteins in nature, for exampleglutamic acid or aspartic acid, and also with phenylacetic acid,methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfoicacid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2-or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid(with the formation of cyclamates), or with other acid organiccompounds, such as ascorbic acid. Pharmaceutically acceptable salts ofcompounds may also be prepared with a pharmaceutically acceptablecation. Suitable pharmaceutically acceptable cations are well known tothose skilled in the art and include alkaline, alkaline earth, ammoniumand quaternary ammonium cations. Carbonates or hydrogen carbonates arealso possible.

[0057] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0058] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of TGF-β is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

[0059] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding TGF-β, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encoding TGF-β canbe detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of TGF-β in a sample may also beprepared.

[0060] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal, intradermal andtransdermal), oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration. Oligonucleotides with at least one2′-O-methoxyethyl modification are believed to be particularly usefulfor oral administration.

[0061] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

[0062] Compositions and formulations for oral administration includepowders or granules, suspensions or solutions in water or non-aqueousmedia, capsules, sachets or tablets. Thickeners, flavoring agents,diluents, emulsifiers, dispersing aids or binders may be desirable.

[0063] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0064] Pharmaceutical compositions and/or formulations comprising theoligonucleotides of the present invention may also include penetrationenhancers in order to enhance the alimentary delivery of theoligonucleotides. Penetration enhancers may be classified as belongingto one of five broad categories, i.e., fatty acids, bile salts,chelating agents, surfactants and non-surfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, 8, 91-192; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).One or more penetration enhancers from one or more of these broadcategories may be included.

[0065] Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid,linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651-654). Examples of some presentlypreferred fatty acids are sodium caprate and sodium laurate, used singlyor in combination at concentrations of 0.5 to 5%.

[0066] The physiological roles of bile include the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York,N.Y., 1996, pages 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus, the term“bile salt” includes any of the naturally occurring components of bileas well as any of their synthetic derivatives. A presently preferredbile salt is chenodeoxycholic acid (CDCA) (Sigma Chemical Company, St.Louis, Mo.), generally used at concentrations of 0.5 to 2%.

[0067] Complex formulations comprising one or more penetration enhancersmay be used. For example, bile salts may be used in combination withfatty acids to make complex formulations. Preferred combinations includeCDCA combined with sodium caprate or sodium laurate (generally 0.5 to5%).

[0068] Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 92-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; Buur et al., J.Control Rel., 1990, 14, 43-51). Chelating agents have the addedadvantage of also serving as DNase inhibitors.

[0069] Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:2,92-191); and perfluorochemical emulsions, such as FC-43 (Takahashi etal., J. Pharm. Pharmacol., 1988, 40, 252-257).

[0070] Non-surfactants include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:2,92-191); and non-steroidal anti-inflammatory agents such as diclofenacsodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm.Pharmacol., 1987, 39, 621-626).

[0071] As used herein, “carrier compound” refers to a nucleic acid, oranalog thereof, which is inert (i.e., does not possess biologicalactivity per se) but is recognized as a nucleic acid by in vivoprocesses that reduce the bioavailability of a nucleic acid havingbiological activity by, for example, degrading the biologically activenucleic acid or promoting its removal from circulation. Thecoadministration of a nucleic acid and a carrier compound, typicallywith an excess of the latter substance, can result in a substantialreduction of the amount of nucleic acid recovered in the liver, kidneyor other extracirculatory reservoirs, presumably due to competitionbetween the carrier compound and the nucleic acid for a common receptor.For example, the recovery of a partially phosphorothioatedoligonucleotide in hepatic tissue is reduced when it is coadministeredwith polyinosinic acid, dextran sulfate, polycytidic acid or4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0072] In contrast to a carrier compound, a “pharmaceutically acceptablecarrier” (excipient) is a pharmaceutically acceptable solvent,suspending agent or any other pharmacologically inert vehicle fordelivering one or more nucleic acids to an animal. The pharmaceuticallyacceptable carrier may be liquid or solid and is selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, etc., when combined with a nucleic acid andthe other components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, but are not limited to,binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate,etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.); disintegrates (e.g., starch, sodium starchglycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate,etc.). Sustained release oral delivery systems and/or enteric coatingsfor orally administered dosage forms are described in U.S. Pat. Nos.4,704,295; 4,556,552; 4,309,406; and 4,309,404.

[0073] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional compatiblepharmaceutically-active materials such as, e.g., antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the composition of present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the invention.

[0074] Regardless of the method by which the antisense compounds of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of thecompounds and/or to target the compounds to a particular organ, tissueor cell type. Colloidal dispersion systems include, but are not limitedto, macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes and lipid:oligonucleotide complexes ofuncharacterized structure. A preferred colloidal dispersion system is aplurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layer(s) made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech., 1995, 6, 698-708).

[0075] Certain embodiments of the invention provide for liposomes andother compositions containing (a) one or more antisense compounds and(b) one or more other chemotherapeutic agents which function by anon-antisense mechanism. Examples of such chemotherapeutic agentsinclude, but are not limited to, anticancer drugs such as daunorubicin,dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard,chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine(5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine,etoposide, teniposide, cisplatin and diethylstilbestrol (DES). See,generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkowet al., eds., 1987, Rahway, N.J., pp. 1206-1228. Anti-inflammatorydrugs, including but not limited to nonsteroidal anti-inflammatory drugsand corticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pp. 2499-2506 and 46-49, respectively. Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0076] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Two or more combinedcompounds may be used together or sequentially.

[0077] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0078] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0079] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-Alkoxy Amidites

[0080] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds. Oligonucleotidescontaining 5-methyl-2′-deoxycytidine (5-Me—C) nucleotides weresynthesized according to published methods (Sanghvi, et. al., NucleicAcids Research, 1993, 21, 3197-3203] using commercially availablephosphoramidites (Glen Research, Sterling Va. or ChemGenes, NeedhamMass.).

[0081] 2′-Fluoro Amidites

[0082] 2′-Fluorodeoxyadenosine Amidites

[0083] 2′-fluoro oligonucleotides were synthesized as describedpreviously by Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841 andU.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0084] 2′-Fluorodeoxyguanosine

[0085] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyrylarabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0086] 2′-Fluorouridine

[0087] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0088] 2′-Fluorodeoxycytidine

[0089] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0090] 2′-O-(2-Methoxyethyl) modified amidites

[0091] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

[0092] 2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0093] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 hours) to give a solid that was crushed to a light tanpowder (57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions or purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.

[0094] 2′-O-Methoxyethyl-5-methyluridine

[0095] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/Acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

[0096] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0097] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0098]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0099] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by tlc by first quenching the tlc sample with the additionof MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane (4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

[0100]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0101] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 hours using an overhead stirrer.POCl₃ was added dropwise, over a 30 minute period, to the stirredsolution maintained at 0-10° C., and the resulting mixture stirred foran additional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

[0102] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0103] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0104]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0105] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, tlc showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0106]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0107]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

Example 2

[0108] Oligonucleotide Synthesis

[0109] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0110] Phosphorothioates (P═S) are synthesized as per the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 seconds and was followed by the capping step.After cleavage from the CPG column and deblocking in concentratedammonium hydroxide at 55° C. (18 hr), the oligonucleotides were purifiedby precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

[0111] Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0112] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0113] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0114] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0115] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0116] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0117] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0118] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0119] Oligonucleoside Synthesis

[0120] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0121] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0122] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0123] PNA Synthesis

[0124] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0125] Synthesis of Chimeric Oligonucleotides

[0126] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0127] [2′-O-Me]-[2′-deoxy]-[2′-O-Me]Chimeric PhosphorothioateOligonucleotides

[0128] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 Ammonia/Ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hours at roomtemperature is then done to deprotect all bases and sample was againlyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for24 hours at room temperature to deprotect the 2′ positions. The reactionis then quenched with 1M TEAA and the sample is then reduced to ½ volumeby rotovac before being desalted on a G25 size exclusion column. Theoligo recovered is then analyzed spectrophotometrically for yield andfor purity by capillary electrophoresis and by mass spectrometry.

[0129] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0130] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0131] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0132] [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0133] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0134] Oligonucleotide Isolation

[0135] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides were purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0136] Oligonucleotide Synthesis—96 Well Plate Format

[0137] Oligonucleotides are synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages are afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages are generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites are purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0138] Oligonucleotides are cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product is thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0139] Oligonucleotide Analysis—96 Well Plate Format

[0140] The concentration of oligonucleotide in each well is assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products is evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACEJ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACEJ 5000, ABI 270). Base and backbone composition isconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates are diluted from the master plateusing single and multi-channel robotic pipettors. Plates are judged tobe acceptable if at least 85% of the compounds on the plate are at least85% full length.

Example 9

[0141] Cell Culture and Oligonucleotide Treatment

[0142] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR, RNAse protection assay(RPA) or Northern blot analysis. The following four human cell types areprovided for illustrative purposes, but other cell types can beroutinely used.

[0143] T-24 cells:

[0144] The transitional cell bladder carcinoma cell line T-24 isobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells are routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells are routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells are seeded into 96-well plates (Falcon-Primaria #3872)at a density of 7000 cells/well for use in RT-PCR analysis.

[0145] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0146] A549 Cells:

[0147] The human lung carcinoma cell line A549 is obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells areroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells are routinely passaged by trypsinization anddilution when they reached 90% confluence.

[0148] NHDF Cells:

[0149] Human neonatal dermal fibroblast (NHDF) are obtained from theClonetics Corporation (Walkersville Md.). NHDFs are routinely maintainedin Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells are maintained for upto 10 passages as recommended by the supplier.

[0150] HEK Cells:

[0151] Human embryonic keratinocytes (HEK) are obtained from theClonetics Corporation (Walkersville Md.). HEKs are routinely maintainedin Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells are routinelymaintained for up to 10 passages as recommended by the supplier.

[0152] Treatment with Antisense Compounds:

[0153] When cells reached 80% confluency, they are treated witholigonucleotide. For cells grown in 96-well plates, wells are washedonce with 200 μL OPTI-MEMJ-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEMJ-1 containing 3.75 μg/mL LIPOFECTINJ(Gibco BRL) and the desired oligonucleotide at a final concentration of150 nM. After 4 hours of treatment, the medium is replaced with freshmedium. Cells are harvested 16 hours after oligonucleotide treatment.

Example 10

[0154] Analysis of Oligonucleotide Inhibition of TGF-62 Expression

[0155] Antisense modulation of TGF-β expression can be assayed in avariety of ways known in the art. For example, TGF-β mRNA levels can bequantitated by Northern blot analysis, RNAse protection assay (RPA),competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR).RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are taught in, for example, Ausubel, et al.,Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons,Inc., 1993, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3. Northern blot analysis isroutine in the art and is taught in, for example, Ausubel, et al.,Current Protocols in Molecular Biology, Volume 1, John Wiley & Sons,Inc., 1996, pp. 4.2.1-4.2.9. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISMJ7700 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.Other methods of PCR are also known in the art.

[0156] TGF-β protein levels can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA, flow cytometry or fluorescence-activated cellsorting (FACS). Antibodies directed to TGF-β can be identified andobtained from a variety of sources, such as PharMingen Inc., San DiegoCalif., or can be prepared via conventional antibody generation methods.Methods for preparation of polyclonal antisera are taught in, forexample, Ausubel, et al., Current Protocols in Molecular Biology, Volume2, John Wiley & Sons, Inc., 1997, pp. 11.12.1-11.12.9. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, et al.,Current Protocols in Molecular Biology, Volume 2, John Wiley & Sons,Inc., 1997, pp. 11.4.1-11.11.5.

[0157] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, et al., Current Protocols in MolecularBiology, Volume 2, John Wiley & Sons, Inc., 1998, pp. 10.16.1-10.16.11.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, et al., Current Protocols in MolecularBiology, Volume 2, John Wiley & Sons, Inc., 1997, pp. 10.8.1-10.8.21.Enzyme-linked immunosorbent assays (ELISA) are standard in the art andcan be found at, for example, Ausubel, et al., Current Protocols inMolecular Biology, Volume 2, John Wiley & Sons, Inc., 1991, pp.11.2.1-11.2.22.

Example 11

[0158] Poly(A)+ mRNA Isolation

[0159] Poly(A)+ mRNA is isolated according to Miura et al., Clin. Chem.,1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation aretaught in, for example, Ausubel, et al., Current Protocols in MolecularBiology, Volume 1, John Wiley & Sons, Inc., 1993, pp. 4.5.1-4.5.3.Briefly, for cells grown on 96-well plates, growth medium is removedfrom the cells and each well is washed with 200 μL cold PBS. 60 μL lysisbuffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mMvanadyl-ribonucleoside complex) is added to each well, the plate isgently agitated and then incubated at room temperature for five minutes.55 μL of lysate is transferred to Oligo d(T) coated 96-well plates (AGCTInc., Irvine Calif.). Plates are incubated for 60 minutes at roomtemperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HClpH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate isblotted on paper towels to remove excess wash buffer and then air-driedfor 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheatedto 70° C. is added to each well, the plate is incubated on a 90° C. hotplate for 5 minutes, and the eluate is then transferred to a fresh96-well plate.

[0160] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0161] Total RNA Isolation

[0162] Total mRNA is isolated using an RNEASY 96J kit and bufferspurchased from Qiagen Inc. (Valencia Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium is removed from the cells and each well iswashed with 200 μL cold PBS. 100 μL Buffer RLT is added to each well andthe plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol isthen added to each well and the contents mixed by pipetting three timesup and down. The samples are then transferred to the RNEASY 96J wellplate attached to a QIAVACJ manifold fitted with a waste collection trayand attached to a vacuum source. Vacuum is applied for 15 seconds. 1 mLof Buffer RW1 is added to each well of the RNEASY 96J plate and thevacuum again applied for 15 seconds. 1 mL of Buffer RPE is then added toeach well of the RNEASY 96J plate and the vacuum applied for a period of15 seconds. The Buffer RPE wash is then repeated and the vacuum isapplied for an additional 10 minutes. The plate is then removed from theQIAVACJ manifold and blotted dry on paper towels. The plate is thenre-attached to the QIAVACJ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA is then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step is repeated with an additional60 μL water.

Example 13

[0163] Real-Time Quantitative PCR Analysis of TGF-62 mRNA Levels

[0164] Quantitation of TGF-β mRNA levels is determined by real-timequantitative PCR using the ABI PRISMJ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE or FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISMJ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0165] PCR reagents are obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions are carried out by adding 25 μL PCRcocktail (1×TAQMANJ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLDJ, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction is carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLDJ, 40 cycles of a two-step PCR protocol arecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

Example 14

[0166] Antisense Inhibition of Murine TGF-β1

[0167] Antisense oligonucleotides were designed to hybridize to themouse TGF-β1 nucleic acid sequence, using published sequence information(GenBank accession No AJ009862; Locus name MMU009862, provided herein asSEQ ID NO: 1). All oligonucleotides have phosphorothioate backbones andare 2′-methoxyethyl (2′-MOE) gapmers. TABLE 1 Antisense oligonucleotidestargeted to mouse TGF-β1 SITE on Nucleotide sequence¹ TARGET ISIS #(5′→3′) SEQUENCE² SEQ ID NO. 105193 TGTCTGGAGGATCCGCGGCG 49 2 105194TGCTCCTTTGCCGGCTCCCA 149 3 105195 CGAGACAGCGCAGTGCCAAG 325 4 105196GGCTCCCGAGGGCTGGTCCG 435 5 105197 GCAGGAGTCGCGGTGAGGCT 696 6 105198AAAGGTGGGATGCGGAGGCC 801 7 105199 CAGTAGCCGCAGCCCCGAGG 875 8 105200AGTCCCGCGGCTGGCCTCCC 937 9 105201 GGCTTCGATGCGCTTCCGTT 992 10 105202GGCGGTACCTCCCCCTGGCT 1057 11 105203 CGCCTGCCACCCGGTCGCGG 1119 12 105204GTCCACCATTAGCACGCGGG 1193 13 105205 GGCACTGCTTCCCGAATGTC 1282 14 105206GTCAGCAGCCGGTTACCAAG 1411 15 105207 AGTGAGCGCTGAATCGAAAG 1515 16 105208GATGGTGCCCAGGTCGCCCC 1598 17 105209 AGGAGCAGGAAGGGCCGGTT 1627 18 105210TCCGGTGCCGTGAGCTGTGC 1680 19 105211 GCCCTTGGGCTCGTGGATCC 1796 20 105212CGCCCGGGTTGTGTTGGTTG 1896 21 105213 GGCTTGCGACCCACGTAGTA 1969 22 105214GGCGGGGCTTCAGCTGCACT 2030 23 110409 CGCCCGGGTTGTGCTGGTTG 1896 24 1 basemismatch to 105212 110410 GTGCTCCCATTGAAAGCCGG 1193 25 8 base mismatchto 105204

[0168]¹Emboldened residues, 2′-methoxyethoxy-residues (others are2′-deoxy-). All C residues, including 2′-MOE and 2′-deoxy residues, are5-methyl-cytosines.

[0169]²Position of first nucleotide at the target site on GenBankaccession No AJ009862; Locus name MMU009862, provided herein as SEQ IDNO: 1).

[0170] The antisense compounds in the table above were screened byNorthern blot at 200 nM oligonucleotide concentration in mouse bEND3endothelial cells (see Montesano et al., Cell, 1990, 62, 435, andStepkowski et al., J. Immunol., 1994, 153, 5336). Cells were treatedwith oligonucleotide (200 nM) and 10 μg/ml of Lipofectin (LifeTechnologies, Inc., Gaithersburg, Md.) for 4 hours. Cells were thenwashed and allowed to recover for a further 24 hr. RNA was isolated andTGF-β1 mRNA expression was measured by Northern blotting. The gels werestripped and reprobed for expression of a housekeeping gene (G3PDH) toconfirm equal loading. TGF-β1 levels are expressed as a percent ofcontrol activity, normalized to G3PDH. Results are shown in Table 2.TABLE 2 Antisense inhibition of mouse TGF-β1 % of Control ISIS #Activity % Inhibition SEQ ID NO. 105193 6 94 2 105194 3 97 3 105195 2080 4 105196 8 92 5 105198 19 81 7 105199 69 31 8 105200 18 82 9 10520186 14 10 105202 209 — 11 105203 160 — 12 105204 47 53 13 105205 12 88 14105206 11 89 15 105207 31 69 16 105208 148 — 17 105209 20 80 18 105211148 — 20 105212 16 84 21 105213 9 91 22 105214 10 90 23

[0171] Oligonucleotides ISIS 105193, 105194, 105195, 105196, 105198,105200, 105204, 105205, 105206, 105207, 105209, 105212, 105213 and105214 gave greater than 50% inhibition of TGF-β1 mRNA in thisexperiment and are preferred.

Example 15

[0172] Dose Response of Antisense Oligonucleotides Targeted to MurineTGF-β1

[0173] bEND.3 cells were treated with oligonucleotides at variousconcentrations with 15 μg/ml Lipofectin for 4 hours, then washed andallowed to recover for 24 hours. TGF-β1 mRNA levels were determined byNorthern blot analysis and normalized to G3PDH levels. Results are shownin Table 3. TABLE 3 Dose response of antisense oligonucleotides targetedto murine TGF-β1 % of Oligonucleotide Dose (nM) Control % Inhib.Lipofectin 100 ISIS 105195 25 47 53 50 35 65 100 25 75 200 18 82 300 892 ISIS 105199 25 115 — 50 126 — 100 125 — 200 103 — ISIS 105204 25 3169 50 22 78 100 16 84 200 11 89 300 11 89 ISIS 105212 25 43 57 50 29 71100 26 74 200 18 82 300 24 76 ISIS 105214 25 30 70 50 17 83 100 17 83200 11 89 300 14 86

[0174] ISIS 105195, 105204, 105212 and 105214 had IC50s below 25 nM inthis experiment and are preferred.

Example 16

[0175] “Humanized” Mouse TGF-β1 Antisense Oligonucleotide

[0176] It was determined by BLAST analysis (Altschul SF et al., J. Mol.Biol. 1990, 215, 403-10) that ISIS 105204, designed to target mouseTGF-β1, has only a single mismatch to the human TGF-β1 gene target, and,except for the 5′-most base on the oligonucleotide, is complementary toa site beginning at nucleotide 1167 on the human target (GenBankaccession no. X02812; locus name HSTGFB1; Derynck, R., et al., 1985,Nature 316, 701-705). An oligonucleotide (TTCCACCATTAGCACGCGGG; ISIS113849; SEQ ID NO: 26) was designed and synthesized which was a completematch to the human target sequence at this site. This compound is aphosphorothioate backbone with 2′-MOE nucleotides shown in bold. All Cresidues are 5-methyl C.

Example 17

[0177] Efficacy of ISIS 105204 in Rat Kidney Cells

[0178] ISIS 105204, designed to target mouse TGF-β1, was tested in ratNRK kidney cells (available from American Type Culture Collection,Manassas Va.). This oligonucleotide has 100% complementarity to the ratTGF-β1 sequence (GenBank accession no.X52498; locus name RNTGFB1,provided herein as SEQ ID NO: 27). A dose response is shown in Table 4.ISIS 105195, which is targeted to a region of the mouse TGF-β1 sequencewhich shares only 9 of 20 nucleobases with the rat sequence, is shownfor comparison. TABLE 4 Dose response of antisense oligonucleotidestargeted to mouse TGF-β1 in rat NRK cells % of Dose Control % ISIS #(nM) activity Inhibition SEQ ID NO Lipofectin 100  — 105195  4 100 115 — 200 98  2 300 97  3 105204 13  50 56 44 100 50 50 200 39 61

Example 18

[0179] Effect of Antisense Inhibition of TGF-β1 on Fibrotic Scarring

[0180] A model for fibrosis has been developed in which osmotic pumpsare implanted subcutaneously in rats. Normally the pump becomesencapsulated by fibrotic scar tissue. The effect of antisense inhibitionof TGF-β1 on scarring can be analyzed and quantitated.

[0181] 2 ml Alzet osmotic pumps (Alza corporation, Palo Alto, Calif.)were implanted subcutaneously on the back of female Sprague Dawley rats.Four rats per experimental group were implanted with pumps containingPBS, 5 mg of TGF-β1 antisense oligonucleotide and 5 mg of an eight basemismatch control oligonucleotide, ISIS 110410. After 3 weeks theencapsulation tissue surrounding the pump was removed, weighed, snapfrozen, and evaluated for TGF-β1 mRNA by Northern blot analysis or RNAseprotection assay using the rCK3b template (Pharmingen, San Diego Calif.)and by immunohistochemistry. For the latter, formalin fixed, paraffinembedded tissues were stained with Masson's Trichrome Stain for Collagenand immunochemical localization of oligonucleotide. Frozen tissues wereantibody-stained for TGF-β1 (antibody from Santa Cruz Biotechnologies,Santa Cruz, Calif.), and EDA Fibronectin (antibody from HarlanBioproducts, Sussex, England). The antibodies were detected withsecondary reagents directly conjugated to HRP and DAB (brown) was usedas the substrate.

[0182] TGF-β1 expression in the scar tissue was reduced by greater than50% after 28-day treatment with ISIS 105204 oligonucleotide dose 15mg/kg; and to greater than 30% with a dose of 5 mg/kg), as measured byNorthern blot analysis of TGF-β1 mRNA levels. This is shown in Table 5.TABLE 5 Effect of ISIS 105204 on TGF-β1 expression in rat scar tissue %of % Oligonucleotide control inhibition SEQ ID NO Saline control 100  —ISIS 110410 (8-base 83 17 25 mismatch of 105204) ISIS 105204 44 56 13

[0183] Immunohistochemical staining showed that TGF-β1 proteinexpression is reduced in scar tissue from mice treated with ISIS 105204.Levels of collagen and fibronectin, which are markers for fibrosis, werealso reduced in scar tissue from these mice. Staining also showed adecrease in the number of CD18 positive cells.

Example 19

[0184] Antisense Oligonucleotides Targeted to Human TGF-β1

[0185] Antisense oligonucleotides were designed to hybridize to thehuman TGF-β1 nucleic acid sequence, using published sequenceinformation; Derynck, R., et al., 1985, Nature 316, 701-705; GenBankaccession number X02812; locus name HSTGFB1, incorporated herein as SEQID NO: 28. Oligonucleotides have phosphorothioate backbones and are2′MOE gapmers. Sequences are shown in Table 6. TABLE 6 Antisenseoligonucleotides targeted to human TGF-β SITE on Nucleotide sequence¹TARGET ISIS # (5′→3′) SEQUENCE² SEQ ID NO: 104978 CGACTCCTTCCTCCGCTCCG113 29 104979 CTCGTCCCTCCTCCCGCTCC 209 30 104980 AAGTCCTGCCTCCTCGCGGG317 31 104981 AAGGGTCTAGGATGCGCGGG 531 32 104982 CTCAGGGAGAAGGGCGCAGT692 33 104983 GCACTGCCGAGAGCGCGAAC 802 34 104984 GTAGCAGCAGCGGCAGCAGC862 35 104985 ATGGCCTCGATGCGCTTCCG 968 36 104986 GCGTAGTAGTCGGCCTCAGG1136 37 104987 ACCACTGCCGCACAACTCCG 1447 38 104988 TCGGCGGCCGGTAGTGAACC1557 39 104989 GAAGTTGGCATGGTAGCCCT 1788 40 104990 GGCGCCCGGGTTATGCTGGT1875 41 104991 CTCCACCTTGGGCTTGCGGC 1956 42 104992 AATGACACAGAGATCCGCAG2155 43 104993 TAGATCTAACTACAGTAGTG 2305 44 104994 CGCCTGGCCTGAACTACTAT2525 45 104995 CCCAGGCTGGTCTCAAATGC 2609 46

[0186] Olignucleotides were screened in 293T human kidney cells at aconcentration of 200 nM with 10 μg/ml of Lipofectin for a period of fourhours. Cells were washed and allowed to recover for a further 24 hr. Atthis point RNA was isolated and TGF-β1 mRNA levels were determined byRibonuclease Protection Assay (RPA) using the hCK-3 template(Pharmingen, San Diego, Calif.) according to the manufacturer'sinstructions. TGF-β1 mRNA levels were normalized to GAPDH and expressedas a percentage of untreated control. Results are shown in Table 7.TABLE 7 Antisense Inhibition of Human TGF-β1 % of Control % ISIS #Activity Inhibition SEQ ID NO: 104978 150  — 29 104979 94  6 30 10498082 18 31 104981 86 14 32 104982 94  6 33 104983 52 48 34 104985 59 41 36104986 59 41 37 104987 63 37 38 104988 71 29 39 104989 86 14 40 10499057 43 41 104991 52 48 42 104992 47 53 43 104993 84 16 44 104994 60 40 45104995 64 36 46 105204 23 77 13

[0187] In this experiment ISIS 104983, 104985, 104986, 104990, 104991,104992, 104994 and 105204 gave at least 40% inhibition of human TGF-β1mRNA and are preferred. ISIS 104992 and 105204 gave over 50% inhibition.

Example 20

[0188] Dose Responses of Antisense Oligonucleotides Targeted to HumanTGF-β1

[0189] ISIS 113849, 105204, 110410 (8 base mismatch of 105204) and104992 were tested at 50, 100 and 200 nM for ability to inhibit TGF-β1mRNA levels. Results are shown in Table 8. TABLE 8 Dose response ofoligonucleotides targeted to human TGF-β1 % of Control SEQ ID ISIS #Dose (nM) Activity % Inhibition NO: 104992  50 65 35 43 100 68 32 200 93 7 105204  50 31 69 13 100 16 84 200 23 77 110410  50 85 15 25 100 64 36200 50 50 113849  50 29 71 26 100 24 76 200 36 64

[0190] ISIS 105204 and 113849 had IC50s below 50 nM in this experiment.These oligonucleotides were found to have little effect on TGF-β2 orTGF-β3 mRNA levels.

Example 21

[0191] Antisense Compounds Targeted to Murine TGF-β2

[0192] Antisense oligonucleotides were designed to hybridize to themouse TGF-β2 nucleic acid sequence, using published sequence informationfrom GenBank accession number X57413; Miller, D. A., et al., Mol.Endocrinol. 1989, 3, 1108-1114; locud name MMTGFB2, incorporated hereinas SEQ ID NO: 47. The oligonucleotides are shown in Table 9. TABLE 9Antisense sequences targeted to murine TGF-β2 SITE on Nucleotidesquence¹ TARGET ISIS # (5′→3′) SEQUENCE² SEQ ID NO. 104996GCCGGCAGTTTCAGCAGCTC 34 48 104997 CTCGCACCCTTCCCTAGCTT 259 49 104998TTTCTTGCTCCAGGCGGCCA 362 50 104999 GAGCAGGCGGCGAGGATCCC 493 51 105000GCCCTGCCTTCCACACGTGT 671 52 105001 GTGCGGAGTGGCTGATCTGA 830 53 105002AAAATGCAACGCGTTCCCAA 1016 54 105003 CCGGGACCAGATGCAGGAGC 1247 55 105004TCCGGCTTGCCTTCTCCTGC 1451 56 105005 GGGTTTTGCAAGCGGAAGAC 1668 57 105006CGATGTAGCGCTGGGTGGGA 1754 58 105007 GGTCTTCCCACTGGTTTTTT 2032 59 105008AAGCTTCGGGATTTATGGTG 2321 60 105009 ACCGTGATTTTCGTGTCCTG 2478 61 105010GCGGGCTGGAAACAATACGT 2854 62 105011 CCCCTGGCTTATTTGAGTTC 3075 63 105012ACCGGCTTGCTTAAACTGGC 3297 64 105013 CAGCCACTTGACGGTCAAAA 3352 65 105014ATGGACCCAGGTAGCTCATG 3753 66 105015 CACCCGCCACATGACTCACA 3874 67 105016TACACCCCATGAGCACCAAA 4097 68

[0193] The oligonucleotides shown in Table 9 were screened for theability to inhibit mouse TGF-β2 mRNA expression by ribonucleaseprotection assay (RPA) in mouse R6+/+ fibroblast cells. Cells weretreated with oligonucleotide (200 nM) and 10 μg/ml of Lipofectin (LifeTechnologies, Inc.) for 4 hours. Cells were then washed and allowed torecover for a further 24 hours. RNA was isolated and TGF-β2 mRNAexpression was measured by RPA using the mCK3b template (Pharmingen,Inc., San Diego Calif.) according to manufacturer's directions. Resultswere normalized to GAPDH and expressed as a percent of RNA levels inuntreated control cells. Results are shown in Table 10. TABLE 10Antisense inhibition of mouse TGF-β2 mRNA expression % of control ISIS #activity % inhibition SEQ ID NO: 104996 83 17 48 104997 79 21 49 10499870 30 50 104999 90 10 51 105000 64 36 52 105001 37 63 53 105002 51 49 54105003 28 72 55 105004 52 48 56 105005 77 23 57 105006 53 47 58 10500760 40 59 105008 55 45 60 105009 28 72 61 105010 27 73 62 105011 48 52 63105012 35 65 64 105013 40 60 65 105014 43 57 66 105015 64 36 67 10501689 11 68

[0194] IS 105001, 105002, 105003, 105004, 105006, 105008, 105009,105010, 105011, 105012, 105013 and 105014 gave at least about 45%inhibition of TGF-β2 mRNA expression in this experiment and arepreferred. Of these, ISIS 105003, 105009 and 105010 gave at least 70%inhibition.

[0195] Interestingly, it was found that oligonucleotides that reducedTGF-β2 also reduced TGF-β3 mRNA levels. This is shown in Table 11. TABLE11 Common inhibition of murine TGF-β2 and TGF-β3 by antisenseoligonucleotides targeted to murine TGF-β2 % inhibition % inhibitionISIS # of TGF-β2 of TGF-β3 SEQ ID NO: 104996 17  3 48 104997 21 11 49104998 30 — 50 104999 10 14 51 105000 36 16 52 105001 63 48 53 105002 4923 54 105003 72 67 55 105004 48 49 56 105005 23 20 57 105006 47 60 58105007 40 29 59 105008 45 23 60 105009 72 55 61 105010 73 49 62 10501152 57 63 105012 65 42 64 105013 60 55 65 105014 57 43 66 105015 36 52 67105016 11 12 68

Example 22

[0196] Reduction in Peritoneal Adhesions by Antisense Inhibition ofTGF-β1

[0197] The surface of the peritoneal cavity and the enclosed organs arecoated with a layer of mesothelial cells that are easily damaged byinjury or infection. Following injury (surgery, for example), adhesionsform which cause permanent scarring. This scarring can result in bowelobstruction, pain, and/or female infertility. A rat model for peritonealadhesions has been developed (Williams et al., 1992, J. Surg. Res. 52,65-70). Animal models have demonstrated that TGF-β promotes theformation of postoperative pelvic adhesions.

[0198] In these experiments bilateral uterine injuries were created in250 gm Sprague Dawley rats by cautery, scraping and crushing. Rats thenreceived 5 mg (20 mg/kg) of an antisense oligonucleotide (ISIS 105204),5 mg of a scrambled control oligonucleotide (ISIS 110410) or 1 mL ofsaline vehicle via intraperitoneal injection.

[0199] Uterine adhesions were then graded by masked evaluators using aclinical scale of 0-3 on days 3, 7 and 14 after injury.

[0200] In order to localize the target tissue of the antisenseoligonucleotides, an additional group of rats were injected with areporter oligonucleotide and biopsies were perfomed on the uterus, liverand kidney of the treated animals. The tissues were then fixed and thereporter oligonucleotide was immunolocalized with a specific antibody.The reporter oligonucleotide was concentrated heavily in the area ofuterine injury at 2 hours and persisted in uterine cells at 72 hoursindicating that the oligonucleotide does localize to the injured area.

[0201] A single dose of the antisense oligonucleotide (ISIS 105204) toTGF-β1 significantly reduced the severity of peritoneal adhesions, froma mean of 3.0 for control animals to 1.2 for antisense treated animals.A scrambled control oligonucleotide (ISIS 110410) gave a mean adhesionscore of 2.4 over the entire study.

Example 23

[0202] Effect of Antisense Inhibition of TGF-β1 on Lung Fibrosis

[0203] A model of lung fibrosis has been developed using bleomycin toinduce pulmonary fibrosis in mice. Wild, J S, S N Giri et al., 1996,Exp. Lung Res. 22, 375-391. Mice receive an intratracheal dose ofbleomycin (0.125U/mouse) or saline, followed by treatment with antisenseoligonucleotide (i.p.) over 2 weeks. Mice were treated with ISIS 105204or 110410. RNA was isolated from lungs and TGF-β1 mRNA levels weredetermined for mice treated with saline or bleomycin alone, saline orbleomycin plus ISIS 105204, and bleomycin plus the scrambled controlISIS 110410. Results are shown in Table 12. These studies showed asignificant reduction of bleomycin-induced lung hydroxyproline content,prolyl hydroxylase and lipid peroxidation. Lung histopathology showedfibrotic lesions to be reduced in bleomycin treated animals receivingthe TGF-β1 oligonucleotide compared to saline or mismatch treatedanimals. Also, RPA (ribonuclease protection assay) analysis revealed a45% reduction in TGF-β1 RNA in animals treated with ISIS 105204. TABLE12 Effect of antisense inhibition of TGF-β1 on lung fibrosis Treatment %of control % inhibition Saline 100  — Saline + 105204 81 19 Bleomycin 7030 Bleomycin + 110410 73 27 Bleomycin + 105204 37 63

Example 24

[0204] Effect of Antisense Inhibition of TGF-β1 on Conjunctival Scarring

[0205] Animal models for a variety of fibrotic diseases and conditionsexist. Conjunctival scarring is a major predictor of visual prognosis ina variety of eye conditions, including post-surgical healing. Forexample, the most common cause of failure of glaucoma filtration surgeryis scarring at the bleb and sclerostomy sites. A model of conjunctivalscarring in the mouse eye has been developed to investigate potentialdeterminants, modes of prevention and treatments for conjunctivalscarring. Reichel et al., 1998, Br. J. Ophthalmol. 82, 1072-1077. Thismodel is used to evaluate the effects of locally or systemicallydelivered antisense to TGF-β on conjunctival scarring. Alternatively,antisense compounds can be administered at the time of trabeculectomyfiltration surgery.

[0206] Study #1

[0207] Animals were anesthetized, and the general glaucoma filtrationtrabeculectomy procedure was followed. A conjunctival flap was raisedand a viscoelastic solution (e.g., Healon) was injected into theanterior chamber. A paracentesis stab incision was made using a 75Beaver blade into the anterior chamber. A sclerotomy was performedthrough the paracentesis incision using a membrane punch and aperipheral iridectomy was done through the sclerostomy. The conjunctivalflap was repositioned and closed with suture in two layers.Oligonucleotide solution (100 μl of 40 μM in the case of rabbits, lessin mouse or rat) was injected into the bleb by tunneling a 30 gaugeneedle through the conjunctiva adjacent to the bleb. Animals weresacrificed 24 hours after treatment and eyes were fixed and examinedhistologically for collagen, fibronectin, and immunohistochemically forTGF-β.

[0208] In this study, Balb-c mice, a highly inbred strain of mice usedto produce monoclonal antibodies, were randomly allocated to one of fivetreatment groups; subconjunctival injection (5 μl) of 25 μg or 12.5 μgof either a TGF-β1 antisense oligonucleotide (ISIS 105204) or ascrambled control oligonucleotide (ISIS 110410), or the carrier salinecontrol. Cellular distribution of oligonucleotide in glaucoma surgerywas assessed following subconjunctival administration of a reporteroligonucleotide into the filtration bleb immediately after surgery inNZW rabbits. Mice and rabbits were assessed clinically and enucleatedeyes were analyzed at set time intervals histologically.

[0209] At days 3 and 7 mouse eyes (n=4) showed significantly reducedwhite cell infiltration and collagen fibril deposition in the TGF-β1oligonucleotide treated groups compared to controls. There was also asignificant decrease in localization of fibroblasts and elastin relatedfibers on days 3,7 and 14 in groups treated with the TGF-β1 antisenseoligonucleotide.

[0210] At 7 days mouse eyes (4 eyes/treatment group) showedsignificantly reduced (p<0.05) conjunctival scar formation in the TGF-β1treated animals as compared to the control group.

[0211] Study #2

[0212] Forty-eight New Zealand White rabbits (Charles River UK Ltd;2-2.4 kg, 12-14 weeks old) were used following an acclimatization periodof around 5 days. All rabbits underwent glaucoma drainage surgery withone of 6 treatments, which were randomly assigned. Of these, 18 rabbitswere sacrificed at 14 days post surgery and the rest were observed until0.30 days. The study was performed as a randomized, controlled studywith masked observers. Animals were randomly assigned to 6 treatmentgroups as shown below. TABLE 13 SEQ No. No. Oligonucleotide ID animalsanimals Group (OGN) and target NO Dose Volume 30 days 14 days 1 ISIS105204 13 100 μg 100 μl 5 3 (TGF-β1) 2 ISIS 123285 69 100 μg 100 μl 5 3(TGF-β2) 3 ISIS 123787 70 100 μg 100 μl 5 3 (TGF-βRII) 4 ISIS 124189 71100 μg 100 μl 5 3 (CTGF) 5 Missense (ISIS 25 100 μg 100 μl 5 3 10410) 6PBS — — 100 μl 5 3

[0213] ISIS 123285 has the sequence 5′-CCGTGACCAGATGCAGGATC-3′. ISIS123787 has the sequence 5′-GGCCAGGGAGCTGCCCAGCT-3′. ISIS 124189 has thesequence 5′-GCCAGAAAGCTCAAACTTGA-3′. These are all targeted to murinesequences. In all three sequences, all internucleoside linkages arephosphorothioates, all cytosines are replaced with 5-methylcytosine, andpositions 1-5 and 16-20 are substituted with 2′-MOE.

[0214] The oligonucleotides were administered immediately pre- andpostoperatively (i.e. on Day 0) to the operated eye of each rabbit bysubconjunctival injection. A 25G needle was placed on the same site ineach eye 5 mm behind the limbus at the nasal margin of the superiorrectus muscle, such that a visible bleb was formed in the supranasalquadrant of each eye. The contralateral eye was used as a control.

[0215] The method described by Cordeiro et al. (Invest. Opthalmol. Vis.Sci. 38:1639-1646, 1997; Cordeiro et al., Invest. Opthalmol. Vis. Sci.40:2225-2234, 1999) was used. All rabbits underwent filtration surgeryto the left eye only. A partial thickness 8-0 silk corneal tractionsuture (Ethicon, Edinburgh, Scotland) was placed superiorly and the eyepulled down. A formix based conjunctival flap was raised following whicha blunt dissection of subconjunctival space was performed to a distanceof 15 mm behind the limbus.

[0216] An MVR blade was used to make a partial thickness scleralincision 4 mm behind the limbus and a scleral tunnel to the cornealstroma was fashioned. A 22 G/25 mm Venflon 2 intravenous cannula waspassed through a scleral tunnel anteriorally until the cannula needlewas visible in the clear cornea. Entry into the anterior chamber wasmade with the cannula needle which was then withdrawn as the cannula wasadvanced to the mid-pupillary area. The cannula was trimmed and bevelledat its scleral end so that it protruded 1 mm from the insertion pointand a 10-0 nulon suture was used to fix the tube to the scleral surface.The conjunctival incision was closed with two interrupted sutures and acentral mattress-type 10-0 nulon suture on a B/V 100-4 needle (Ethicon)to give a water-tight closure. One drop of atropine sulfate 1% andbetnesol N ointment was instilled at the end of surgery.

[0217] All animals were checked ophthalmologically at baseline and everyday for the first 3 days after surgery and thereafter every third orfourth day until Day 30, as described below. Ophthalmological assessmentincluded intraocular pressure, bleb size, bleb vascularity, anteriorchamber depth and anterior chamber activity.

[0218] Measurement of intraocular pressure in both eyes was made usingthe Mentor tonopen. This was performed after topical installation of0.4% benoxinate HCl local anesthetic. Three recordings per eye were madeper time point and a mean reading was documented.

[0219] Bleb width, height and length was measured and bleb area (widthand length) was calculated. Measurements were made with a microsurgicalcaliper in mm.

[0220] Bleb and conjunctival vascularity was performed by dividing theconjunctival area into quadrants: superior, nasal and temporal. Eachquadrant was then assessed and recorded using color photography. Gradingwas performed as follows: 0=avascular, +1=normal vascularity,+2=hyperemic, +3=very hyperemic. Avascularity was also assessed andgraded binomially as follows: 1=presence of avascularity in any area ofthe eye, 0=no vascularity.

[0221] Anterior chamber depth was assessed subjectively, graded andrecorded as either deep (=+2), shallow (=+1) or flat (═O).

[0222] Anterior chamber activity (inflammation) was assessed by slitlamp photography and graded as follows: 0=no cells, +1=cells present,+2=fibrin formation, +3=a hypopyon.

[0223] A general macroscopic description was recorded on the injectedarea in terms of complications such as lid edema, chemosis, hemorrhageand corneal toxicity.

[0224] The primary efficacy endpoint was taken as bleb survival. Blebfailure was defined as the appearance of a flat, vascularized, scarredbleb in association with a deep anterior chamber. Kaplan-Meier and logrank statistics were used to compare treatment groups. The multivariateanalysis of variance (ANOVA) was used to compare differences betweentreatments and effects of time and treatment, using the SPSS package andthe Bonferroni correction. Bleb area and height were analyzed using therepeated measures procedure by the Generalized Linear Model (SPSS). Thisallowed comparison of treatment groups over the whole study period usingthe tests of between-subjects. Anterior chamber depth and activity wereassessed using the General Linear Model, as described above. Analysis ofconjunctival vascularity changes was performed using GLM statistics asdescribed above for superior, temporal and nasal quadrants. Avascularitywas assessed using Pearson's Chi-Squared test to compare treatmentgroups.

[0225] At the beginning of the study, all rabbits receiving treatmentwith Group 3 antisense oligonucleotide developed endopthalmitis within 3days of surgery. A very heavy growth (confluent) of Staphylococcusaureus was isolated from these cases which were sacrificed in accordancewith the protocol and Home Office regulations. As the cause of theinfection was isolated, and the causative batch of oligonucleotidesidentified, a new treatment group was substituted and treated with afresh batch of stringently tested Group 3 antisense oligonucleotide.There was one case of death following administration of the anestheticbut prior to surgery (rabbit 42).

[0226] A Kaplan-Meier bleb survival curve was constructed and is shownin FIG. 1, with the mean survival in each treatment group shown in Table14. Survival was prolonged in TGF-β2 and TGF-βIIR antisenseoligonucleotide groups (mean survival of 19.4 and 16.5 days,respectively) compared to the TGF-β1, CTGF and control groups. Treatmentwith the TGF-β2 antisense oligonucleotide significantly prolonged blebsurvival compared to TGF-β1 (log rank p=0.0009), CTGF (p=0.0042),missense (p=0.0072) oligonucleotide and PBS control (p=0.0035) treatmentgroups. Compared to PBS control, TGF-β2 antisense oligonucleotideincreased bleb survival by 5.68 days, and TGF-βIIR antisenseoligonucleotide increased bleb survival by 2.78 days. TABLE 14 TGF-β2TGF-βIIR TGF-β1 CTGF Missense PBS Group OGN OGN OGN OGN OGN control Mean19.4 16.5 14.88 14.2 14.37 13.72 survival (days) Median 17.0 17.0 16.014.0 14.0 14.0 survival (days)

[0227] Analysis of intraocular pressure showed no significant differencebetween treatment groups and at any time point. Bleb area and heightwere analyzed using the repeated measures procedure by the GeneralizedLinear Model (GLM). Bleb area and height were analyzed using therepeated measures procedure by the Generalized Linear Model (SPSS).Comparison of treatment groups over the whole study period revealed nosignificant difference between treatment groups with respect to blebarea or bleb height. Anterior chamber depth was assessed using GLM asdescribed above. TGF-β2 oligonucleotide treated eyes had shalloweranterior chamber depth compared to the other treatments. Comparisonbetween treatment groups showed a significant difference (p=0.034). FIG.2 shows the mean grade of anterior chamber depth in the operated eyeover the study period. GLM was also used to compare anterior chamberinflammation between treatment groups, and was found to be significantlydifferent (p=0.02). Vascularity was graded in each quadrant and analyzedusing GLM statistics as described above. Comparison of treatment groupsfor each quadrant showed no significant difference throughout the studyperiod. However, at day 1, the PBS control group was significantly morevascular than the other treatment groups in the superior and temporalquadrants.

[0228] The TGF-β2 antisense oligonucleotide significantly prolonged blebsurvival by 5.68 days after perioperative application in glaucomafiltration surgery in the rabbit. Bleb survival was pre-defined as theprimary efficacy endpoint of the study. Although bleb survival was alsoprolonged with the TGF-βIIR antisense oligonucleotide group, this didnot reach statistical significance when compared to control. Treatmentwith the TGF-β2 antisense oligonucleotide significantly prolonged blebsurvival compared to TGF-β1 (log rank p=0.0009), CTGF (p=0.0042),missense (p=0.0072) and PBS control (p=0.0035) treatment groups.

[0229] No difference was noted between treatment groups with regard tointraocular pressure. This may be due to a breakdown in theblood-aqueous barrier producing destabilization of intraocular pressure.Evidence of increased aqueous outflow through the filtration site in 6B1treatment groups compared to controls may also be obtained frommeasurements of anterior chamber depth and bleb morphology. Thus,shallow anterior chambers and higher and larger blebs indicate improvedoutflow. Shallow anterior chambers were observed for longer periods oftime in the TGF-β2 antisense oligonucleotide treatment group.

[0230] No difference between treatment groups was recorded in relationto conjunctival vascularity and anterior chamber activity over the wholestudy period. This parameter is important in assessing the safety of theinjected substances. If local tolerance was present, it would be seenclinically as increased vascular injection and uveitis (increasedanterior chamber activity). It is interesting to note, however, that atday 1, the PBS control group was significantly more vascular than theother treatment groups, suggesting that all test substances were in factvery well-tolerated and perhaps even anti-inflammatory compared to thePBS control.

[0231] The results show that TGF-β2 antisense oligonucleotide treatmentis effective in reducing the conjunctival scarring response followingglaucoma filtration surgery in a model of aggressive scarring. Despitethe relatively small numbers of animals in the treatment group, the factthat statistical significance in bleb survival was achieved, shows thepotency of antisense oligonucleotides to TGF-β2. In addition, theoligonucleotide appears to be well tolerated in vivo, with no evidenceof adverse reactions. These results show that TGF-β2 is an effective andsage anti-scarring therapeutic agent.

[0232] The cellular profile suggested that TGF-β1 oligonucleotidedelayed the development of the wound healing response.Immunohistochemical staining with an antibody specific for the reporteroligonucleotide in rabbit eyes revealed intense and localized stainingof the TGF-β1 oligonucleotide to fibroblasts, epithelial cells andmacrophages in the sclera and conjunctiva at the surgical site.

Example 25

[0233] Effect of Antisense Inhibition of TGF-β1 on Inflammation-HumanSkin Xenograft Model in the SCID Mouse.

[0234] Another model used to investigate the processes of inflammationand scarring involves the use of SCID mice transplanted with human skin.SCID mice lack an enzyme necessary to fashion an immune system and cantherefore be converted into a model of the human immune system wheninjected with human cells or tissues. In these experiments human skin (2cm²) from various surgical procedures (breast reductions or neonatalforeskin) or from cadavers was transplanted onto the side of SCID micewith sutures or surgical staples. After four to six weeks, the mice werebled and tested for Ig to ensure the SCID lineage. After 8 to 10 weeks,the xenograft skin was treated with antisense oligonucleotide, ISIS105204, SEQ ID NO: 13) in a cream formulation at 48, 24, and 4 hoursprior to the injection of 4000U of tumor necrosis factor-alpha (TNF-α).Levels of TGF-β protein were then assayed in the epidermis and dermis ofthe xenograft skin by immunohistochemical staining 24 hours after TNF-αinjection. Levels were reported as a percentage of the area showingpositive staining for the presence of TGF-β protein.

[0235] In the epidermis, 3% of the area showed positive staining aftertreatment with TGF-β antisense oligonucleotide relative to basal levelsof 50% and levels of 37% for the placebo cream group. This data wasshown to be statistically significant (P=0.0001).

[0236] In the dermis, TGF-β levels were below 0.5% with basal levels at2.5% and placebo cream group levels of 2%.

1 71 1 2094 DNA Mus musculus CDS (868)...(2040) 1 cgccgccgcc gccgcccttcgcgccccagg ccgtccccct cctcctcccg ccgcggatcc 60 tccagacagc caggcccccggccggggcag gggggacgcc ccttcggggc acccccggct 120 ctgagccgca ctcggagtcggcctccgctg ggagccggca aaggagcagc cgaggagccg 180 tccgaggccc cagagtctgagaccagccgc cgccgcaggg aggaggggga ggaggagtgg 240 gaggagggac gagctggttgagagaagagg aaaaaagttt tgagactttt ccgctgctac 300 tgcaagtcag agacgtggggacttcttggc actgcgctgt ctcgcaagga ggcaggacct 360 gaggactcca gacagccctgctcaccgtcg tggacactcg atcgctaccc ggcgttcctc 420 agacgcccct attccggaccagccctcggg agccacaaac cccgcctccc gcgaagactt 480 caccccaaag ctggggcgcaccccttgcac gccgccctcc ccccagcctg cctcttgagt 540 ccctcgcatc ccaggaccctctctcccccg agaggcagat ctccctcgga cctgctggca 600 gtagctcccc tatttaagaacacccacttt tggatctcag agagcgctca tctcgatttt 660 taccctggtg gtatactgagacaccttggt gtcagagcct caccgcgact cctgctgctt 720 tctccctcaa cctcaaattattcaggacta tcacctacct ttccttggga gaccccaccc 780 cacaagccct gcaggggcggggcctccgca tcccaccttt gccgagggtt cccgctctcc 840 gaagtgccgt ggggcgccgcctccccc atg ccg ccc tcg ggg ctg cgg cta ctg 894 Met Pro Pro Ser Gly LeuArg Leu Leu 1 5 ccg ctt ctg ctc cca ctc ccg tgg ctt cta gtg ctg acg cccggg agg 942 Pro Leu Leu Leu Pro Leu Pro Trp Leu Leu Val Leu Thr Pro GlyArg 10 15 20 25 cca gcc gcg gga ctc tcc acc tgc aag acc atc gac atg gagctg gtg 990 Pro Ala Ala Gly Leu Ser Thr Cys Lys Thr Ile Asp Met Glu LeuVal 30 35 40 aaa cgg aag cgc atc gaa gcc atc cgt ggc cag atc ctg tcc aaacta 1038 Lys Arg Lys Arg Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu45 50 55 agg ctc gcc agt ccc cca agc cag ggg gag gta ccg ccc ggc ccg ctg1086 Arg Leu Ala Ser Pro Pro Ser Gln Gly Glu Val Pro Pro Gly Pro Leu 6065 70 ccc gag gcg gtg ctc gct ttg tac aac agc acc cgc gac cgg gtg gca1134 Pro Glu Ala Val Leu Ala Leu Tyr Asn Ser Thr Arg Asp Arg Val Ala 7580 85 ggc gag agc gcc gac cca gag ccg gag ccc gaa gcg gac tac tat gct1182 Gly Glu Ser Ala Asp Pro Glu Pro Glu Pro Glu Ala Asp Tyr Tyr Ala 9095 100 105 aaa gag gtc acc cgc gtg cta atg gtg gac cgc aac aac gcc atctat 1230 Lys Glu Val Thr Arg Val Leu Met Val Asp Arg Asn Asn Ala Ile Tyr110 115 120 gag aaa acc aaa gac atc tca cac agt ata tat atg ttc ttc aatacg 1278 Glu Lys Thr Lys Asp Ile Ser His Ser Ile Tyr Met Phe Phe Asn Thr125 130 135 tca gac att cgg gaa gca gtg ccc gaa ccc cca ttg ctg tcc cgtgca 1326 Ser Asp Ile Arg Glu Ala Val Pro Glu Pro Pro Leu Leu Ser Arg Ala140 145 150 gag ctg cgc ttg cag aga tta aaa tca agt gtg gag caa cat gtggaa 1374 Glu Leu Arg Leu Gln Arg Leu Lys Ser Ser Val Glu Gln His Val Glu155 160 165 ctc tac cag aaa tat agc aac aat tcc tgg cgt tac ctt ggt aaccgg 1422 Leu Tyr Gln Lys Tyr Ser Asn Asn Ser Trp Arg Tyr Leu Gly Asn Arg170 175 180 185 ctg ctg acc ccc act gat acg cct gag tgg ctg tct ttt gacgtc act 1470 Leu Leu Thr Pro Thr Asp Thr Pro Glu Trp Leu Ser Phe Asp ValThr 190 195 200 gga gtt gta cgg cag tgg ctg aac caa gga gac gga ata cagggc ttt 1518 Gly Val Val Arg Gln Trp Leu Asn Gln Gly Asp Gly Ile Gln GlyPhe 205 210 215 cga ttc agc gct cac tgc tct tgt gac agc aaa gat aac aaactc cac 1566 Arg Phe Ser Ala His Cys Ser Cys Asp Ser Lys Asp Asn Lys LeuHis 220 225 230 gtg gaa atc aac ggg atc agc ccc aaa cgt cgg ggc gac ctgggc acc 1614 Val Glu Ile Asn Gly Ile Ser Pro Lys Arg Arg Gly Asp Leu GlyThr 235 240 245 atc cat gac atg aac cgg ccc ttc ctg ctc ctc atg gcc accccc ctg 1662 Ile His Asp Met Asn Arg Pro Phe Leu Leu Leu Met Ala Thr ProLeu 250 255 260 265 gaa agg gcc cag cac ctg cac agc tca cgg cac cgg agagcc ctg gat 1710 Glu Arg Ala Gln His Leu His Ser Ser Arg His Arg Arg AlaLeu Asp 270 275 280 acc aac tat tgc ttc agc tcc aca gag aag aac tgc tgtgtg cgg cag 1758 Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys ValArg Gln 285 290 295 ctg tac att gac ttt agg aag gac ctg ggt tgg aag tggatc cac gag 1806 Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp IleHis Glu 300 305 310 ccc aag ggc tac cat gcc aac ttc tgt ctg gga ccc tgcccc tat att 1854 Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys ProTyr Ile 315 320 325 tgg agc ctg gac aca cag tac agc aag gtc ctt gcc ctctac aac caa 1902 Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu TyrAsn Gln 330 335 340 345 cac aac ccg ggc gct tcg gcg tca ccg tgc tgc gtgccg cag gct ttg 1950 His Asn Pro Gly Ala Ser Ala Ser Pro Cys Cys Val ProGln Ala Leu 350 355 360 gag cca ctg ccc atc gtc tac tac gtg ggt cgc aagccc aag gtg gag 1998 Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys ProLys Val Glu 365 370 375 cag ttg tcc aac atg att gtg cgc tcc tgc aag tgcagc tga 2040 Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser 380 385390 agccccgccc cgccccgccc ctcccggcag gcccggcccc gcccccgccc cgcc 2094 220 DNA Artificial Sequence Antisense Oligonucleotide 2 tgtctggaggatccgcggcg 20 3 20 DNA Artificial Sequence Antisense Oligonucleotide 3tgctcctttg ccggctccca 20 4 20 DNA Artificial Sequence AntisenseOligonucleotide 4 cgagacagcg cagtgccaag 20 5 20 DNA Artificial SequenceAntisense Oligonucleotide 5 ggctcccgag ggctggtccg 20 6 20 DNA ArtificialSequence Antisense Oligonucleotide 6 gcaggagtcg cggtgaggct 20 7 20 DNAArtificial Sequence Antisense Oligonucleotide 7 aaaggtggga tgcggaggcc 208 20 DNA Artificial Sequence Antisense Oligonucleotide 8 cagtagccgcagccccgagg 20 9 20 DNA Artificial Sequence Antisense Oligonucleotide 9agtcccgcgg ctggcctccc 20 10 20 DNA Artificial Sequence AntisenseOligonucleotide 10 ggcttcgatg cgcttccgtt 20 11 20 DNA ArtificialSequence Antisense Oligonucleotide 11 ggcggtacct ccccctggct 20 12 20 DNAArtificial Sequence Antisense Oligonucleotide 12 cgcctgccac ccggtcgcgg20 13 20 DNA Artificial Sequence Antisense Oligonucleotide 13 gtccaccattagcacgcggg 20 14 20 DNA Artificial Sequence Antisense Oligonucleotide 14ggcactgctt cccgaatgtc 20 15 20 DNA Artificial Sequence AntisenseOligonucleotide 15 gtcagcagcc ggttaccaag 20 16 20 DNA ArtificialSequence Antisense Oligonucleotide 16 agtgagcgct gaatcgaaag 20 17 20 DNAArtificial Sequence Antisense Oligonucleotide 17 gatggtgccc aggtcgcccc20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18 aggagcaggaagggccggtt 20 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19tccggtgccg tgagctgtgc 20 20 20 DNA Artificial Sequence AntisenseOligonucleotide 20 gcccttgggc tcgtggatcc 20 21 20 DNA ArtificialSequence Antisense Oligonucleotide 21 cgcccgggtt gtgttggttg 20 22 20 DNAArtificial Sequence Antisense Oligonucleotide 22 ggcttgcgac ccacgtagta20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 ggcggggcttcagctgcact 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24ccacgtagta gacgatgggc 20 25 20 DNA Artificial Sequence AntisenseOligonucleotide 25 gtccaccatt agcacgcggg 20 26 20 DNA ArtificialSequence Antisense Oligonucleotide 26 gtccaccatt agcacgcggg 20 27 1585DNA Rattus norvegicus CDS (413)...(1585) 27 accgcctccc gcaaagacttcaccccaaag ctggggcgca ccccttgcac gccaccctcc 60 ccccagcctg cttcttgagtcccccgcatc ccaggaccct ctctcctctg ggaggccgat 120 ctccctcgga cctgctggcaatagcttcct atttaagaac accccacttt tgggtcccag 180 agagcgctca tctcgatttttatcccggtg gcatactgag acactctggt gtcagagcgt 240 caccgcgact cctgctgctttctccctcaa cctcaaatta ttcaggacta tcacctacct 300 ttccttggga gaccccaccccaccccacaa gccctgcagg ggcggggcct ccgcatccca 360 cctttgcccg gggttcgcgctctccgaagt tccgtggggc gccgcctccc cc atg ccg 418 Met Pro 1 ccc tcg gggctg cgg ctg ctg ccg ctt ctg ctc cca ctc ccg tgg ctt 466 Pro Ser Gly LeuArg Leu Leu Pro Leu Leu Leu Pro Leu Pro Trp Leu 5 10 15 cta gtg ctg acgccc ggg agg cca gcc gcg gga ctc tcc acc tgc aag 514 Leu Val Leu Thr ProGly Arg Pro Ala Ala Gly Leu Ser Thr Cys Lys 20 25 30 acc atc gac atg gagctg gtg aaa cgg aag cgc atc gaa gcc atc cgt 562 Thr Ile Asp Met Glu LeuVal Lys Arg Lys Arg Ile Glu Ala Ile Arg 35 40 45 50 ggc cag atc ctg tccaaa cta agg ctc gcc agt ccc ccg agc cag ggg 610 Gly Gln Ile Leu Ser LysLeu Arg Leu Ala Ser Pro Pro Ser Gln Gly 55 60 65 gag gta ccg ccg ggc ccgctg ccc gag gcg gtg ctc gct ttg tac aac 658 Glu Val Pro Pro Gly Pro LeuPro Glu Ala Val Leu Ala Leu Tyr Asn 70 75 80 agc acc cgc gac cgg gtg gcaggc gag agc gct gac ccg gag ccc gag 706 Ser Thr Arg Asp Arg Val Ala GlyGlu Ser Ala Asp Pro Glu Pro Glu 85 90 95 ccc gag gcg gac tac tac gcc aaagaa gtc acc cgc gtg cta atg gtg 754 Pro Glu Ala Asp Tyr Tyr Ala Lys GluVal Thr Arg Val Leu Met Val 100 105 110 gac cgc aac aac gca atc tat gacaaa acc aaa gac atc aca cac agt 802 Asp Arg Asn Asn Ala Ile Tyr Asp LysThr Lys Asp Ile Thr His Ser 115 120 125 130 ata tat atg ttc ttc aat acgtca gac att cgg gaa gca gtg cca gaa 850 Ile Tyr Met Phe Phe Asn Thr SerAsp Ile Arg Glu Ala Val Pro Glu 135 140 145 ccc cca ttg ctg tcc cgt gcagag ctg cgc ctg cag aga ttc aag tca 898 Pro Pro Leu Leu Ser Arg Ala GluLeu Arg Leu Gln Arg Phe Lys Ser 150 155 160 act gtg gag caa cac gta gaactc tac cag aaa tat agc aac aat tcc 946 Thr Val Glu Gln His Val Glu LeuTyr Gln Lys Tyr Ser Asn Asn Ser 165 170 175 tgg cgt tac ctt ggt aac cggctg ctg acc ccc act gat acg cct gag 994 Trp Arg Tyr Leu Gly Asn Arg LeuLeu Thr Pro Thr Asp Thr Pro Glu 180 185 190 tgg ctg tct ttt gac gtc actgga gtt gtc cgg cag tgg ctg aac caa 1042 Trp Leu Ser Phe Asp Val Thr GlyVal Val Arg Gln Trp Leu Asn Gln 195 200 205 210 gga gac gga ata cag ggcttt cgc ttc agt gct cac tgc tct tgt gac 1090 Gly Asp Gly Ile Gln Gly PheArg Phe Ser Ala His Cys Ser Cys Asp 215 220 225 agc aaa gat aat gta ctccac gtg gaa atc aat ggg atc agt ccc aaa 1138 Ser Lys Asp Asn Val Leu HisVal Glu Ile Asn Gly Ile Ser Pro Lys 230 235 240 cgt cga ggt gac ctg ggcacc atc cat gac atg aac cga ccc ttc ctg 1186 Arg Arg Gly Asp Leu Gly ThrIle His Asp Met Asn Arg Pro Phe Leu 245 250 255 ctc ctc atg gcc acc cccctg gaa agg gct caa cac ctg cac agc tcc 1234 Leu Leu Met Ala Thr Pro LeuGlu Arg Ala Gln His Leu His Ser Ser 260 265 270 agg cac cgg aga gcc ctggat acc aac tac tgc ttc agc tcc aca gag 1282 Arg His Arg Arg Ala Leu AspThr Asn Tyr Cys Phe Ser Ser Thr Glu 275 280 285 290 aag aac tgc tgt gtacgg cag ctg tac att gac ttt agg aag gac ctg 1330 Lys Asn Cys Cys Val ArgGln Leu Tyr Ile Asp Phe Arg Lys Asp Leu 295 300 305 ggt tgg aag tgg atccac gag ccc aag ggc tac cat gcc aac ttc tgt 1378 Gly Trp Lys Trp Ile HisGlu Pro Lys Gly Tyr His Ala Asn Phe Cys 310 315 320 ctg ggg ccc tgc ccctac att tgg agc ctg gac aca cag tac agc aag 1426 Leu Gly Pro Cys Pro TyrIle Trp Ser Leu Asp Thr Gln Tyr Ser Lys 325 330 335 gtc ctt gcc ctc tacaac caa cac aac ccg ggt gct tcc gca tca ccg 1474 Val Leu Ala Leu Tyr AsnGln His Asn Pro Gly Ala Ser Ala Ser Pro 340 345 350 tgc tgc gtg ccg caggct ttg gag cca ctg ccc atc gtc tac tac gtg 1522 Cys Cys Val Pro Gln AlaLeu Glu Pro Leu Pro Ile Val Tyr Tyr Val 355 360 365 370 ggt cgc aag cccaag gtg gag cag ttg tcc aac atg atc gtg cgc tcc 1570 Gly Arg Lys Pro LysVal Glu Gln Leu Ser Asn Met Ile Val Arg Ser 375 380 385 tgc aag tgc agctga 1585 Cys Lys Cys Ser 390 28 2745 DNA Homo sapiens CDS (842)...(2017)28 acctccctcc gcggagcagc cagacagcga gggccccggc cgggggcagg ggggacgccc 60cgtccggggc accccccccg gctctgagcc gcccgcgggg ccggcctcgg cccggagcgg 120aggaaggagt cgccgaggag cagcctgagg ccccagagtc tgagacgagc cgccgccgcc 180cccgccactg cggggaggag ggggaggagg agcgggagga gggacgagct ggtcgggaga 240agaggaaaaa aacttttgag acttttccgt tgccgctggg agccggaggc gcggggacct 300cttggcgcga cgctgccccg cgaggaggca ggacttgggg accccagacc gcctcccttt 360gccgccgggg acgcttgctc cctccctgcc ccctacacgg cgtccctcag gcgcccccat 420tccggaccag ccctcgggag tcgccgaccc ggcctcccgc aaagactttt ccccagacct 480cgggcgcacc ccctgcacgc cgccttcatc cccggcctgt ctcctgagcc cccgcgcatc 540ctagaccctt tctcctccag gagacggatc tctctccgac ctgccacaga tcccctattc 600aagaccaccc accttctggt accagatcgc gcccatctag gttatttccg tgggatactg 660agacaccccc ggtccaagcc tcccctccac cactgcgccc ttctccctga ggagcctcag 720ctttccctcg aggccctcct accttttgcc gggagacccc cagcccctgc aggggcgggg 780cctccccacc acaccagccc tgttcgcgct ctcggcagtg ccggggggcg ccgcctcccc 840 catg ccg ccc tcc ggg ctg cgg ctg ctg ccg ctg ctg cta ccg ctg ctg 889 MetPro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Leu 1 5 10 15tgg cta ctg gtg ctg acg cct ggc ccg ccg gcc gcg gga cta tcc acc 937 TrpLeu Leu Val Leu Thr Pro Gly Pro Pro Ala Ala Gly Leu Ser Thr 20 25 30 tgcaag act atc gac atg gag ctg gtg aag cgg aag cgc atc gag gcc 985 Cys LysThr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala 35 40 45 atc cgcggc cag atc ctg tcc aag ctg cgg ctc gcc agc ccc ccg agc 1033 Ile Arg GlyGln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser 50 55 60 cag ggg gaggtg ccg ccc ggc ccg ctg ccc gag gcc gtg ctc gcc ctg 1081 Gln Gly Glu ValPro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu 65 70 75 80 tac aac agcacc cgc gac cgg gtg gcc ggg gag agt gca gaa ccg gag 1129 Tyr Asn Ser ThrArg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu 85 90 95 ccc gag cct gaggcc gac tac tac gcc aag gag gtc acc cgc gtg cta 1177 Pro Glu Pro Glu AlaAsp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu 100 105 110 atg gtg gaa acccac aac gaa atc tat gac aag ttc aag cag agt aca 1225 Met Val Glu Thr HisAsn Glu Ile Tyr Asp Lys Phe Lys Gln Ser Thr 115 120 125 cac agc ata tatatg ttc ttc aac aca tca gag ctc cga gaa gcg gta 1273 His Ser Ile Tyr MetPhe Phe Asn Thr Ser Glu Leu Arg Glu Ala Val 130 135 140 cct gaa ccc gtgttg ctc tcc cgg gca gag ctg cgt ctg ctg agg agg 1321 Pro Glu Pro Val LeuLeu Ser Arg Ala Glu Leu Arg Leu Leu Arg Arg 145 150 155 160 ctc aag ttaaaa gtg gag cag cac gtg gag ctg tac cag aaa tac agc 1369 Leu Lys Leu LysVal Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser 165 170 175 aac aat tcctgg cga tac ctc agc aac cgg ctg ctg gca ccc agc gac 1417 Asn Asn Ser TrpArg Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp 180 185 190 tcg cca gagtgg tta tct ttt gat gtc acc gga gtt gtg cgg cag tgg 1465 Ser Pro Glu TrpLeu Ser Phe Asp Val Thr Gly Val Val Arg Gln Trp 195 200 205 ttg agc cgtgga ggg gaa att gag ggc ttt cgc ctt agc gcc cac tgc 1513 Leu Ser Arg GlyGly Glu Ile Glu Gly Phe Arg Leu Ser Ala His Cys 210 215 220 tcc tgt gacagc agg gat aac aca ctg caa gtg gac atc aac ggg ttc 1561 Ser Cys Asp SerArg Asp Asn Thr Leu Gln Val Asp Ile Asn Gly Phe 225 230 235 240 act accggc cgc cga ggt gac ctg gcc acc att cat ggc atg aac cgg 1609 Thr Thr GlyArg Arg Gly Asp Leu Ala Thr Ile His Gly Met Asn Arg 245 250 255 cct ttcctg ctt ctc atg gcc acc ccg ctg gag agg gcc cag cat ctg 1657 Pro Phe LeuLeu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu 260 265 270 caa agctcc cgg cac cgc cga gcc ctg gac acc aac tat tgc ttc agc 1705 Gln Ser SerArg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser 275 280 285 tcc acggag aag aac tgc tgc gtg cgg cag ctg tac att gac ttc cgc 1753 Ser Thr GluLys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg 290 295 300 aag gacctc ggc tgg aag tgg atc cac gag ccc aag ggc tac cat gcc 1801 Lys Asp LeuGly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala 305 310 315 320 aacttc tgc ctc ggg ccc tgc ccc tac att tgg agc ctg gac acg cag 1849 Asn PheCys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln 325 330 335 tacagc aag gtc ctg gcc ctg tac aac cag cat aac ccg ggc gcc tcg 1897 Tyr SerLys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser 340 345 350 gcggcg ccg tgc tgc gtg ccg cag gcg ctg gag ccg ctg ccc atc gtg 1945 Ala AlaPro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val 355 360 365 tactac gtg ggc cgc aag ccc aag gtg gag cag ctg tcc aac atg atc 1993 Tyr TyrVal Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile 370 375 380 gtgcgc tcc tgc aag tgc agc tga ggtcccgccc cgccccgccc cgccccggca 2047 ValArg Ser Cys Lys Cys Ser 385 390 ggcccggccc caccccgccc cgcccccgctgccttgccca tgggggctgt atttaaggac 2107 accgtgcccc aagcccacct ggggccccattaaagatgga gagaggactg cggatctctg 2167 tgtcattggg cgcctgcctg gggtctccatccctgacgtt cccccactcc cactccctct 2227 ctctccctct ctgcctcctc ctgcctgtctgcactattcc tttgcccggc atcaaggcac 2287 aggggaccag tggggaacac tactgtagttagatctattt attgagcacc ttgggcactg 2347 ttgaagtgcc ttacattaat gaactcattcagtcaccata gcaacactct gagatggcag 2407 ggactctgat aacacccatt ttaaaggttgaggaaacaag cccagagagg ttaagggagg 2467 agttcctgcc caccaggaac ctgctttagtgggggatagt gaagaagaca ataaaagata 2527 gtagttcagg ccaggcgggg tgctcacgcctgtaatccta gcacttttgg gaggcagaga 2587 tgggaggata cttgaatcca ggcatttgagaccagcctgg gtaacatagt gagaccctat 2647 ctctacaaaa cacttttaaa aaatgtacacctgtggtccc agctactctg gaggctaagg 2707 tgggaggatc acttgatcct gggaggtcaaggctgcag 2745 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29cgactccttc ctccgctccg 20 30 20 DNA Artificial Sequence AntisenseOligonucleotide 30 ctcgtccctc ctcccgctcc 20 31 20 DNA ArtificialSequence Antisense Oligonucleotide 31 aagtcctgcc tcctcgcggg 20 32 20 DNAArtificial Sequence Antisense Oligonucleotide 32 aagggtctag gatgcgcggg20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 ctcagggagaagggcgcagt 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34gcactgccga gagcgcgaac 20 35 20 DNA Artificial Sequence AntisenseOligonucleotide 35 gtagcagcag cggcagcagc 20 36 20 DNA ArtificialSequence Antisense Oligonucleotide 36 atggcctcga tgcgcttccg 20 37 20 DNAArtificial Sequence Antisense Oligonucleotide 37 gcgtagtagt cggcctcagg20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 accactgccgcacaactccg 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39tcggcggccg gtagtgaacc 20 40 20 DNA Artificial Sequence AntisenseOligonucleotide 40 gaagttggca tggtagccct 20 41 20 DNA ArtificialSequence Antisense Oligonucleotide 41 ggcgcccggg ttatgctggt 20 42 20 DNAArtificial Sequence Antisense Oligonucleotide 42 ctccaccttg ggcttgcggc20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 aatgacacagagatccgcag 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44tagatctaac tacagtagtg 20 45 20 DNA Artificial Sequence AntisenseOligonucleotide 45 cgcctggcct gaactactat 20 46 20 DNA ArtificialSequence Antisense Oligonucleotide 46 cccaggctgg tctcaaatgc 20 47 4267DNA Mus musculus CDS (1218)...(2462) 47 ggttatctgc tggcagcagg tttgctcggagcagagctgc tgaaactgcc ggcaggagag 60 cgagtgggag agaaagagag aaggcgctgagagctgagct ctggggcagg cgtcagggat 120 ggagagaagt attagggttt aaagagccattctggagcaa cccatctgcg gagagaagga 180 tcggcagagg tctattttag ggtcgcaagtacctacttac cctaagcgag aaagtgcaac 240 cttggtggaa gctaggagaa gctagggaagggtgcgagtc ccggggcagc ccgcagccaa 300 cgcgcccagg aggcggtgtt gttccacaggggttaaggag gtggccgatc gctgtcgccc 360 ttggccgcct ggagcaagaa aaggaggatctgaaggaccg agctggaggc tggccctctt 420 tgcaggcagc agcggcggct gcaacgtggagcgacccagc cgggtgtagg ccacagcgcg 480 gccggcagga gcgggatcct cgccgcctgctccggcctct gtggatctcc ggggcggaca 540 gtatcccacc gagtctccga gtgagccgctccggggcgca tctgcctccc cgcggctcgc 600 caggctcgcc ctcggcgcgc gcgcacgcacgcgcgcacac gcgcacacat ccacacgcac 660 actcatccac acacgtgtgg aaggcagggccgagccgctc ggtctttgaa cttctcagtt 720 agagcccggc gcagccccgg ccgccgctcagcgctccccg cggccctgcg tgcctcctgc 780 cagcccccgg accttctcgt ctcgtcccttttggccggag gatcggagtt cagatcagcc 840 actccgcacc gagcctgaca cactgaactccatttcttcc tcttaagttt atttctactt 900 cagagccact caccctctcc cttccaggagaaaaaaaaaa caaacctttc ttactcctta 960 aagtgagaga ttcccccccc accccgccccagcatcgcat attaatatct ccacgttggg 1020 aacgcgttgc attttctttt ttaaaggaatcccagccagg aacgtttttc tattgggcat 1080 taactttcga ctgctttgca aaagtttcgtattaaagaac aactctacct gaccgctctg 1140 agaattacta gtttcttttt tatatatattttttcttact ttaaataaca acatcaacgt 1200 ttcttccttt taaaaac atg cac tac tgtgtg ctg agc acc ttt ttg ctc 1250 Met His Tyr Cys Val Leu Ser Thr Phe LeuLeu 1 5 10 ctg cat ctg gtc ccg gtg gcg ctc agt ctg tct acc tgc agc accctc 1298 Leu His Leu Val Pro Val Ala Leu Ser Leu Ser Thr Cys Ser Thr Leu15 20 25 gac atg gat cag ttt atg cgc aag agg atc gag gcc atc cgc ggg cag1346 Asp Met Asp Gln Phe Met Arg Lys Arg Ile Glu Ala Ile Arg Gly Gln 3035 40 atc ctg agc aag ctg aag ctc acc agc ccc ccg gaa gac tat ccg gag1394 Ile Leu Ser Lys Leu Lys Leu Thr Ser Pro Pro Glu Asp Tyr Pro Glu 4550 55 ccg gat gag gtc ccc ccg gag gtg att tcc atc tac aac agt acc agg1442 Pro Asp Glu Val Pro Pro Glu Val Ile Ser Ile Tyr Asn Ser Thr Arg 6065 70 75 gac tta ctg cag gag aag gca agc cgg agg gca gcc gcc tgc gag cgc1490 Asp Leu Leu Gln Glu Lys Ala Ser Arg Arg Ala Ala Ala Cys Glu Arg 8085 90 gag cgg agc gag cag gag tac tac gcc aag gag gtt tat aaa atc gac1538 Glu Arg Ser Glu Gln Glu Tyr Tyr Ala Lys Glu Val Tyr Lys Ile Asp 95100 105 atg ccg tcc cac ctc ccc tcc gaa aat gcc atc ccg ccc act ttc tac1586 Met Pro Ser His Leu Pro Ser Glu Asn Ala Ile Pro Pro Thr Phe Tyr 110115 120 aga ccc tac ttc aga atc gtc cgc ttt gat gtc tca aca atg gag aaa1634 Arg Pro Tyr Phe Arg Ile Val Arg Phe Asp Val Ser Thr Met Glu Lys 125130 135 aat gct tcg aat ctg gtg aag gca gag ttc agg gtc ttc cgc ttg caa1682 Asn Ala Ser Asn Leu Val Lys Ala Glu Phe Arg Val Phe Arg Leu Gln 140145 150 155 aac ccc aaa gcc aga gtg gcc gag cag cgg att gaa ctg tat cagatc 1730 Asn Pro Lys Ala Arg Val Ala Glu Gln Arg Ile Glu Leu Tyr Gln Ile160 165 170 ctt aaa tcc aaa gac tta aca tct ccc acc cag cgc tac atc gatagc 1778 Leu Lys Ser Lys Asp Leu Thr Ser Pro Thr Gln Arg Tyr Ile Asp Ser175 180 185 aag gtt gtg aaa acc aga gcg gag ggt gaa tgg ctc tcc ttc gacgtg 1826 Lys Val Val Lys Thr Arg Ala Glu Gly Glu Trp Leu Ser Phe Asp Val190 195 200 aca gac gct gtg cag gag tgg ctt cac cac aaa gac agg aac ctgggg 1874 Thr Asp Ala Val Gln Glu Trp Leu His His Lys Asp Arg Asn Leu Gly205 210 215 ttt aaa ata agt tta cac tgc ccc tgc tgt acc ttc gtg ccg tctaat 1922 Phe Lys Ile Ser Leu His Cys Pro Cys Cys Thr Phe Val Pro Ser Asn220 225 230 235 aat tac atc atc ccg aat aaa agc gaa gag ctc gag gcg agattt gca 1970 Asn Tyr Ile Ile Pro Asn Lys Ser Glu Glu Leu Glu Ala Arg PheAla 240 245 250 ggt att gat ggc acc tct aca tat gcc agt ggt gat cag aaaact ata 2018 Gly Ile Asp Gly Thr Ser Thr Tyr Ala Ser Gly Asp Gln Lys ThrIle 255 260 265 aag tcc act agg aaa aaa acc agt ggg aag acc cca cat ctcctg cta 2066 Lys Ser Thr Arg Lys Lys Thr Ser Gly Lys Thr Pro His Leu LeuLeu 270 275 280 atg ttg ttg ccc tcc tac aga ctg gag tca caa cag tcc agccgg cgg 2114 Met Leu Leu Pro Ser Tyr Arg Leu Glu Ser Gln Gln Ser Ser ArgArg 285 290 295 aag aag cgc gct ttg gat gct gcc tac tgc ttt aga aat gtgcag gat 2162 Lys Lys Arg Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val GlnAsp 300 305 310 315 aat tgc tgc ctt cgc cct ctt tac att gat ttt aag agggat ctt gga 2210 Asn Cys Cys Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg AspLeu Gly 320 325 330 tgg aaa tgg atc cat gaa ccc aaa ggg tac aat gct aacttc tgt gct 2258 Trp Lys Trp Ile His Glu Pro Lys Gly Tyr Asn Ala Asn PheCys Ala 335 340 345 ggg gca tgc cca tat cta tgg agt tca gac act caa cacacc aaa gtc 2306 Gly Ala Cys Pro Tyr Leu Trp Ser Ser Asp Thr Gln His ThrLys Val 350 355 360 ctc agc ctg tac aac acc ata aat ccc gaa gct tcc gcttcc cct tgc 2354 Leu Ser Leu Tyr Asn Thr Ile Asn Pro Glu Ala Ser Ala SerPro Cys 365 370 375 tgt gtg tcc cag gat ctg gaa cca ctg acc att ctc tattac att gga 2402 Cys Val Ser Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr TyrIle Gly 380 385 390 395 aat acg ccc aag atc gaa cag ctt tcc aat atg attgtc aag tct tgt 2450 Asn Thr Pro Lys Ile Glu Gln Leu Ser Asn Met Ile ValLys Ser Cys 400 405 410 aaa tgc agc taa agtccttggg aaagccagga cacgaaaatcacggtgacaa 2502 Lys Cys Ser tgacatataa tgacaacgat gacgaccatg atgtttgtgacaggagggag ggagttttga 2562 ttcatcagtg tttaaaaaaa aaaaaattgg agaaaaaaaatcggtactag ttcaaacatt 2622 ttgcaagctt gtgttctgtt tgttaaaact ggcatctgagattacagcaa caacaaccac 2682 aaaaatggaa ggcgttagtc tgcatctcac ctacttcctaagagacacaa aaagaaaaca 2742 tctttttttt tttaaggaaa aaaataaaca ctggaagaatttgttagtgt taattatgtg 2802 aaaaaaaaaa aacatcaaaa caaaacagga aaatccgttcagtggagttg tacgtattgt 2862 ttccagcccg catttcaccc cacgcctctc ctggttcctctgtattgctc tctgcagtgg 2922 gtgccctccc cgtcccttcc tccaagctaa cagtgggttatttattgtgt gttactatat 2982 aatgaacctt tcattaccct tggaaaacaa aacaggtgtataaatcgaga ccaaatactt 3042 tgccacaaac tcatggatgg cttaaggagt ttgaactcaaataagccagg gggaaggagg 3102 tcatagtgga tgaccccctg tgagttgtta taggactaagcaagtcttct gtggaaaaat 3162 caaagcccca gcaaacacgt gtctgccgaa gcttcatggacgccatatgc ccagaaggcc 3222 tgttaacaaa gaaaacttgg aatcagtggc aatctggaagattttttttt ccttttaatt 3282 gtaaatggtt ctttgccagt ttaagcaagc cggtgaaatgttgacctgtt ttgatatgta 3342 ttgtcagact tttgaccgtg aagtggctgt tgatctacaatacaggtttt tcctttgtct 3402 tggtatatgt aattacatgg atactattaa aatagacgggtctagaagcc agcatgattg 3462 aaaacacact gcagatctgt ttttccaaac tattaaatcgaaacagtaac tactttacat 3522 gtaatgtgta gatcttacca catttttaat attctgtaataatggttatg atttagattg 3582 aacttaaatt tggacttttt tttttaatga tcattcagattgtatatttg tttcctttag 3642 ctggccagta cctttgaata aaacccctag attttgacttgcactacaaa ttcaattttt 3702 tttatatact atcttccctg cctgtatttt atgtattgtccatttaatga catgagctac 3762 ctgggtccat tcctccccca accccagttc cttctattttccaaaagata aaaaccaaag 3822 cccaaaaagc taggtttgag ctccacagtg tttcagccttttctgcgtca gtgtgagtca 3882 tgtggcgggt gagcggtggg gcttctggga tggatggttctgtgtgaaca cagaagttcg 3942 cacaaatgta ggcttagcta gggtttaaga atctcaactcagagtcttag tgactgggct 4002 aggaaaagtt tctttaactc ctatatttat ggactctctttgccgttcaa aagcagacag 4062 ttcaaaggaa gcaccttttt ctttaattgg tttttttggtgctcatgggg tgtattaaaa 4122 gacacacagt ttggttgagt ttttcaaagg gggaaaaagtccaggccagc actcgtcatt 4182 ttattcataa tttcatccat tatttccctg atttcattgaaatacaggtt ttgaaagaca 4242 ttctttgcag gctgattaaa aaaaa 4267 48 20 DNAArtificial Sequence Antisense Oligonucleotide 48 gccggcagtt tcagcagctc20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 ctcgcacccttccctagctt 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50tttcttgctc caggcggcca 20 51 20 DNA Artificial Sequence AntisenseOligonucleotide 51 gagcaggcgg cgaggatccc 20 52 20 DNA ArtificialSequence Antisense Oligonucleotide 52 gccctgcctt ccacacgtgt 20 53 20 DNAArtificial Sequence Antisense Oligonucleotide 53 gtgcggagtg gctgatctga20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 aaaatgcaacgcgttcccaa 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55ccgggaccag atgcaggagc 20 56 20 DNA Artificial Sequence AntisenseOligonucleotide 56 tccggcttgc cttctcctgc 20 57 20 DNA ArtificialSequence Antisense Oligonucleotide 57 gggttttgca agcggaagac 20 58 20 DNAArtificial Sequence Antisense Oligonucleotide 58 cgatgtagcg ctgggtggga20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 ggtcttcccactggtttttt 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60aagcttcggg atttatggtg 20 61 20 DNA Artificial Sequence AntisenseOligonucleotide 61 accgtgattt tcgtgtcctg 20 62 20 DNA ArtificialSequence Antisense Oligonucleotide 62 gcgggctgga aacaatacgt 20 63 20 DNAArtificial Sequence Antisense Oligonucleotide 63 cccctggctt atttgagttc20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 accggcttgcttaaactggc 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65cagccacttc acggtcaaaa 20 66 20 DNA Artificial Sequence AntisenseOligonucleotide 66 atggacccag gtagctcatg 20 67 20 DNA ArtificialSequence Antisense Oligonucleotide 67 cacccgccac atgactcaca 20 68 20 DNAArtificial Sequence Antisense Oligonucleotide 68 tacaccccat gagcaccaaa20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 ccgtgaccagatgcaggatc 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70ggccagggag ctgcccagct 20 71 20 DNA Artificial Sequence AntisenseOligonucleotide 71 gccagaaagc tcaaacttga 20

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targetedto a nucleic acid molecule encoding TGF-β2, wherein said compoundcomprises at least a portion of a sequence selected from the groupconsisting of SEQ ID NO:53, 54, 55, 56, 58, 60, 61, 62, 63, 64, 65, 66or 69, and wherein said compound modulates the expression of TGF-β2. 2.The compound of claim 1 which is an antisense oligonucleotide.
 3. Thecompound of claim 2 wherein the antisense oligonucleotide comprises atleast one modified internucleoside linkage.
 4. The compound of claim 3wherein the modified internucleoside linkage of the antisenseoligonucleotide is a phosphorothioate linkage.
 5. The compound of claim2 wherein the antisense oligonucleotide comprises at least one modifiedsugar moiety.
 6. The compound of claim 5 wherein the modified sugarmoiety of the antisense oligonucleotide is a 2′-O-methoxyethyl sugarmoiety.
 7. The compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified nucleobase.
 8. The compound of claim 7wherein the modified nucleobase of the antisense oligonucleotide is a5-methylcytosine.
 9. The compound of claim 2 wherein the antisenseoligonucleotide is a chimeric oligonucleotide.
 10. A compositioncomprising the compound of claim 1 and a pharmaceutically acceptablecarrier or diluent.
 11. The composition of claim 10 further comprising acolloidal dispersion system.
 12. The composition of claim 10 wherein thecompound is an antisense oligonucleotide.
 13. A method of inhibiting theexpression of TGF-β2 in cells or tissues comprising contacting saidcells or tissues with the compound of claim 1 so that expression ofTGF-β2 is inhibited.
 14. A method of treating an animal having a diseaseor condition associated with TGF-β2 comprising administering to saidanimal a therapeutically or prophylactically effective amount of thecompound of claim 1 so that expression of TGF-β2 is inhibited.
 15. Themethod of claim 14 wherein said disease or condition is inflammation.16. The method of claim 14 wherein said disease or condition is fibrosisor a fibrotic disease or condition.
 17. The method of claim 16, whereinsaid fibrotic disease or condition is fibrotic scarring, peritonealadhesions, lung fibrosis or conjunctival scarring.